Protein-protein interaction as biomarkers

ABSTRACT

The subject invention pertains to materials and methods for the classification of cancers as sensitive or resistant to treatments based on protein-protein interactions, treatment of cancer, identification of biomarkers, identification of protein-protein interaction modulators, and selection of cancer treatments.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. CA119997awarded by the National Institute of Health (NIH). The government hascertain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Pat. No. 6,1540,212,filed Sep. 28, 2011, which is hereby incorporated by reference herein inits entirety, including any figures, tables, nucleic acid sequences,amino acid sequences, and drawings.

BACKGROUND OF THE INVENTION

A better understanding of tumor subsets that benefit from treatment isof critical importance to enable personalized medicine. For this reason,a number of molecular approaches to classify cancer and identifyresponsive subsets are now being tested. Proteomic strategies (whichexamine global patterns of protein expression or phosphorylation) arealso being used to identify subsets of tumors. This includes classicimmunohistochemistry approaches to measure protein expression inparaffin fixed tumor sections, use of phospho-specific antibodies tomeasure specific phosphorylation events on particular proteins,proteomic profiling tools (such as reverse phase protein arrays), andmass spectrometry based approaches. Biomarker systems to measureprotein-protein interaction biomarkers in cancer have lagged behindthese other tools. This is an important missing component of mostbiomarker strategies, as cellular signaling requires proper formation ofsignaling complexes and networks of proteins that act in concert toproduce a physiological signal.

It is known that oncoproteins, such as epidermal growth factor receptor(EGFR) or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS),produce a signaling network comprised of a defined set of molecules thatlead to “oncogene addiction” and cell transformation. A betterunderstanding of the oncoprotein signaling network could uncover noveltherapeutic targets or therapeutic strategies and allow “networkmedicine” to become a reality.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods for theidentification of malignancies as suitable for treatment, treatment ofmalignancies, and selection of treatments (prophylactic and/ortherapeutic interventions). The oncoprotein signaling network canprovide biomarkers categorized by protein-protein interactions (PPI)that carry information to guide clinical decision making. The presentinvention includes materials and methods for determining theresponsiveness of a malignancy to a therapeutic intervention, such as amodulator (inhibitor or inducer) of the PPI, by measuring PPI in asample of the cancer.

Aspects of the invention include a method for assessing the sensitivityof a malignancy to a potential treatment; a method for treating amalignancy in a subject that is predetermined to be sensitive to a PPImodulator based on a PPI profile obtained from a sample of themalignancy; a kit for quantitatively detecting the proximity of targetbinding partners within a cancer PPI network; a kit for quantitativelydetecting the proximity of target binding partners within a tertiaryinteraction (an interaction of three or more target binding partners); amethod for the identification of a biomarker; and a method foridentifying an agent as a PPI modulator.

Assays such as proximity ligation assays (PLAs) can be used toquantitatively measure defined PPI in the cancer sample and determinePPI expression patterns to establish a relationship to clinical outcome.PLA technology is capable of detecting single protein events such asprotein interactions (e.g., protein dimerization) and modifications(e.g., protein phosphorylation) in tissue and cell samples prepared formicroscopy. Two interacting proteins in a complex are identified withtwo primary antibodies (of different species) specific for the proteinone primary antibody for each interacting protein (Soderberg O. et al.,“Direct observation of individual endogenous protein complexes in situby proximity ligation”, Nat. Methods., 2006 December; 3(12):995-1000;and Jarvius M. et al., “In situ detection of phosphorylatedplatelet-derived growth factor receptor beta using a generalizedproximity ligation method”, Mol. Cell. Proteomics, 2007 September;6(9):1500-9, which are each incorporated herein by reference in itsentirety”). Species-specific secondary antibodies (referred to herein as“PLA probes” or “proximity probes”) each have a unique short nucleicacid strand attached to it. When the PLA probes are in close proximity(e.g., less than 40 nm), the nucleic acid strands (through the additionof two circle-forming oligonucleotides) become ligated together byenzymatic ligation and form a circle that functions as a template foramplification (see FIG. 2A). Because the distance of the nucleic acidstrands is small, only proteins that interact will allow ligation. Afterthe amplification reaction, the resulting rolling circular amplification(RCA) serves as a target for labeled (e.g., fluorescently labeled)complimentary oligonucleotide probes (see FIG. 2B), which allow fordetection (e.g., visualization) and quantification of the product (e.g.,using a florescent microscope). This technique greatly amplifies thesignal for each antigen recognition event. In this way, the signal isamplified by generating an amplified nucleic acid surrogate of thebinding partners (proteins) in sufficient proximity to produce a signal.The signal from each detected pair of proximity probes can bevisualized, e.g., as a fluorescent dot, and quantified (counted) andassigned to a specific subcellular location (localization of signal)based on microscopy images.

The methods and kits of the invention make use of PLAs to measure PPI incell samples, e.g., cancer samples. The PPIs may be binary (having twoprotein binding partners) or tertiary (having three or more proteinbinding partners). Proteins within a tertiary interaction may be boundto one or more proteins within the PPI. For measurement of binaryinteractions, oligonucleotide probes for one type of signal (forexample, one color, such as red). For measurement of tertiaryinteractions, a second signal that is discernable from the first signalis required for detection of the second interaction. Instead of twoprimary antibodies, three primary antibodies can be used, each of adifferent species (mouse, rabbit, and goat, for example). For example,in measuring a tertiary interaction between protein binding partnersrepresented as A, B, and C, two separate PLAs are carried out, with afirst signal (e.g., red signal) representing interaction between A andB, and a second signal (distinguishable from the first signal)representing interaction between B and C (e.g., green signal).Optionally, for visualization, the images can be constructed showingeach signal (e.g., red and green signals) with overlay.

PPI measurements can be taken of virtually any oncoprotein and itsbinding partner(s). In some embodiments, the oncoprotein comprisesepidermal growth factor receptor (EGFR). As EGFR is a key kinase in lungcancer, in some embodiments, the oncoprotein comprises EGFR and thecancer sample is a lung cancer sample.

One aspect of the invention concerns a method for assessing thesensitivity of a malignancy to a treatment based on PPI. The method forassessing the sensitivity of a malignancy to a treatment comprisescomparing a protein-protein interaction (PPI) profile obtained from asample of the malignancy to a reference PPI profile. The sample PPIprofile represents the abundance of target binding partners that are inproximity to each other within the sample. A negative result (lack ofPPI) in a sample would indicate a lack of sensitivity to the PPImodulator and be useful clinically to avoid giving patients unnecessaryPPI modulator therapy. A positive result (presence of PPI) wouldindicate that the malignancy is potentially sensitive to the PPImodulator and allow the clinician to give the PPI modulator therapy tothose patients who would be most likely to benefit. As a specificexample, the method may be used for determining whether there is no EGFsignaling in a sample such as spleen tissue, and this kind of a negativeresult can be useful clinically to avoid giving patients unnecessary EGFreceptor inhibitor therapy, or alternatively be used to give the therapyto those patients who would be most likely to benefit.

PPI profiles (e.g., sample PPI profile, and reference PPI profile) mayeach be expressed as a value representative of the abundance of targetbinding partners in proximity to each other within the sample. Thesample PPI profile and reference PPI profile may be expressed by anymethod useful for comparison purposes, such as a numeric value, score,cutoff (threshold), or other expression. For example, a negative resultin which a PPI profile in a sample does not reach the cutoff would beuseful clinically to avoid giving patients unnecessary PPI modulatortherapy. A positive result in which a PPI profile in a sample is at orabove the cutoff would indicate potential sensitivity and allow theclinician to give the PPI modulator therapy to those patients who wouldbe most likely to benefit.

In some embodiments of the methods and kits of the invention, the targetPPI is that of a known cancer signaling network. Binding members of atarget PPI may include protein products of oncogenes or tumor suppressorgenes, for example. In some embodiments, the sample PPI profilerepresents the abundance of target binding partners that are inproximity to each other within the sample, wherein at least one of thetarget binding partners is selected from among EGFR, ALK, MET, IGFR,Src, ErbB3, Mig6, Grb2, Sts1, p85, and Hsp90.

In some embodiments of the methods and kits of the invention, the targetbinding partners are selected from among EGFR and ErbB3; EGFR and Grb2;EGFR and Mig6; EGFR and Sts1, EGFR and Src; EGFR and Hsp90; ErbB3 andp85; ALK and EML4; MET and Gab1; IGFR and IRS; Hsp90 and Cdc37; ALK andShc1; EGFR and Shc1.

In some embodiments of the methods and kits of the invention, the PPIcomprises a tyrosine kinase, such as the human tyrosine kinases listedin Tables 3 and 4. In some embodiments, the PPI comprises an interactionbetween the binding partners listed in Table 5 (human tyrosine kinaseinteractions).

PLAs can be used to generate biomarkers against receptor tyrosinekinases (RTKs). RTKs are important proteins in cancer and highly“druggable” targets. Approximately twenty different RTK classes havebeen identified thus far. In some embodiments of the methods and kits ofthe invention, at least one of the target binding partners is an RTK. Insome embodiments of the methods and kits of the invention, at least oneof the target binding partners is of an RTK class selected from amongRTK class I (EGF receptor family; ErbB family), RTK class II (insulinreceptor family), RTK class III (PDGF receptor family), RTK class IV(FGF receptor family) RTK class V (VEGF receptors family), RTK class VI(HGF receptor family), RTK class VII (Trk receptor family), RTK classVIII (Eph receptor family), RTK class IX (AXL receptor family), RTKclass X (LTK receptor family), RTK class XI (TIE receptor family), RTKclass XII (ROR receptor family), XIII (DDR receptor family), RTK classXIV (RET receptor family), RTK class XV (KLG receptor family), RTK classXVI (RYK receptor family), and RTK class XVII (MuSK receptor family).

In some embodiments of the methods and kits of the invention, one ormore of the binding partners of the target PPI have one or more sequencemutations that are known to be associated with occurrence of themalignancy. In other embodiments, the binding partners of the target PPIdo not harbor (lack) any sequence mutations known to be associated withoccurrence of the malignancy, or with sensitivity to a treatment such asa PPI modulator. In some embodiments, the binding partners of the targetPPI and the downstream effectors of the target PPI do not harbor anysequence mutations known to be associated with occurrence of themalignancy, or with sensitivity to a treatment such as a PPI modulator.Such sequence aberrations in a subject or in a sample can be detectedusing methods known in the art (e.g., mutation analysis). Typically,abnormalities in nucleic acid sequences are identified by comparison toreference sequence data (sequences of normal cells or cancer cells) ondatabases, such as GenBank and EMBL, and specific data resources such asCancer Gene Census (mutated genes causally implicated in human cancer),COSMIC (Catalogue of Somatic Mutations in Cancer), and CGP ResequencingStudies (somatic mutations from large scale resequencing of genes inhuman cancer). Mutations causing or contributing to cancer may belarge-scale mutations, involving the deletion or addition of a portionof a chromosome, or small-scale mutations, including point mutations,deletions, insertions, which may occur in the promoter region of a geneand affect its expression, may occur in the coding sequence and alterthe stability or function of the gene's protein product.

The sample may be any cell sample potentially harboring the targetprotein(s). For example, a cytology sample may be obtained from a tissueselected from breast, ovaries, esophagus, stomach, colon, rectum, anus,bile duct, brain, endometrium, lung, liver, skin, prostate, kidney,nasopharynx, pancreas, head and neck, kidney, lymphoma, leukemia,cervix, and bladder. The sample may be a solid or non-solid tumorspecimen. The tumor specimen may be a carcinoma. The sample may be a newcancer, recurrent cancer, primary cancer, or metastasized (secondary)cancer.

The sample may be obtained by methods known in the art, such as surgery,biopsy, or from blood (e.g., circulating tumor cells), ascites, orpleural effusion. The sample may be processed using methods known in theart. For example, the sample may be fresh, frozen, or formalin-fixed andparaffin-embedded (FFPE).

Preferably, the treatment against which the sample is being assessed forsensistivity/resistance is a PPI modulator (i.e., a PPI inhibitor or PPIinducer). However, the treatment may be a treatment other than a PPImodulator.

Another aspect of the invention concerns a method for treating amalignancy in a subject, comprising administering a protein-proteininteraction (PPI) modulator to the subject, wherein the subject ispredetermined to be sensitive to the PPI modulator based on a PPIprofile obtained from a sample of the malignancy. In some embodiments,the PPI modulator is an inhibitor of the PPI of the PPI profile (i.e., aPPI inhibitor). In some embodiments, the PPI modulator is an inducer ofthe PPI of the PPI profile (i.e., a PPI inducer).

Another aspect of the invention concerns a method for treating amalignancy in a subject, comprising:

(a) assessing the sensitivity of a malignancy in the subject, comprisingcomparing a protein-protein interaction (PPI) profile obtained from asample of the malignancy to a reference PPI profile; and

(b) administering a PPI modulator to the subject if the malignancy isassessed to be sensitive to the PPI modulator; and withholding the PPImodulator from the subject if the malignancy is assessed to be resistantto the PPI modulator. In some embodiments, the PPI modulator is aninhibitor of the PPI of the PPI profile (i.e., a PPI inhibitor). In someembodiments, the PPI modulator is an inducer of the PPI of the PPIprofile (i.e., a PPI inducer).

The invention includes also includes kits useful for carrying outmethods of the invention, (e.g., methods for the classification ofcancers as sensitive or resistant to treatments based on protein-proteininteractions, treatment of cancer, identification of biomarkers,identification of protein-protein interaction modulators, and selectionof cancer treatments). Thus, one aspect of the invention concerns a kitfor detecting the proximity of target binding partners within a cancerprotein-protein interaction network, comprising a primary antibody to atleast one of the target binding partners, and a proximity probecomprising a secondary antibody (that binds to the primary antibody)with an oligonucleotide conjugated thereto. In prefened embodiments, thekit comprises:

a first primary antibody to a first target binding partner;

a second primary antibody to a second target binding partner;

a first proximity probe comprising a first secondary antibody (thatbinds to the first primary antibody) with an oligonucleotide conjugatedthereto; and

a second proximity probe comprising a second secondary antibody (thatbinds to the second primary antibody) with an oligonucleotide conjugatedthereto, wherein when the oligonucleotides of the first and secondproximity probes are in sufficient proximity to each other, theoligonucleotides of the proximity probes interact in the presencecircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing an amplification product. Optionally, the kit furthercomprises a labeled oligonucleotide probe that hybridizes with theamplification product, allowing detection and quantification of theamplification product (representing the association (close proximity) ofthe target binding partners).

Another aspect of the invention concerns a kit for detecting theproximity of target binding partners within tertiary PPI (a complexhaving three or more protein binding partners), referred to herein asthe “tertiary interaction kit”. The target PPI may be within a cancersignaling network, but is not limited to such applications. The tertiaryinteraction kit can be used to study viruses, for example, in which aviral protein forms an interaction with protein A and protein B in acell, giving rise to a tertiary complex. Identification of this tertiaryPPI may be diagnostic of an active infection or yield importantinformation about the prognosis or predict the correct therapy. Incancer, a tertiary complex may provide important information about thetherapeutic efficacy.

The tertiary interaction kit comprises:

a first primary antibody to a first target binding partner of thetertiary interaction;

a second primary antibody to a second target binding partner of thetertiary interaction;

a third primary antibody to a third target binding partner of thetertiary interaction;

a first proximity probe comprising a first secondary antibody (thatbinds to the first primary antibody) with an oligonucleotide conjugatedthereto; and

a second proximity probe comprising a second secondary antibody (thatbinds to the second primary antibody) with an oligonucleotide conjugatedthereto;

a third proximity probe comprising a third secondary antibody (thatbinds to the third primary antibody) with an oligonucleotide conjugatedthereto;

wherein when the oligonucleotides of the first and second proximityprobes are in sufficient proximity to each other, the oligonucleotidesof the first and second proximity probes interact in the presencecircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing a first amplification product; and

wherein when the oligonucleotides of the second and third proximityprobes are in sufficient proximity to each other, the oligonucleotidesof the second and third proximity probes interact in the presence ofcircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing a second amplification product. Optionally, the kit detectingproximity of binding partners within a tertiary PPI further comprises afirst labeled oligonucleotide probe that hybridizes with the firstamplification product, allowing detection and quantification of thefirst amplification product (representing the association (closeproximity) of the first and second target binding partners), and asecond labeled oligonucleotide probe that hybridizes with the secondamplification product, allowing detection and quantification of thesecond amplification product (representing the association of the secondand third target binding partners). Each primary antibody is of adifferent species (e.g., mouse, rabbit, and goat, for example).Preferably, the label of the first labeled oligonucleotide yields asignal (e.g., a color) that is distinguishable from that of the secondlabeled oligonucleotide (red and green, for example). An additionalprimary antibody, proximity probe, and (optionally) oligonucleotideprobe can be included in the kit for each additional target bindingpartner within the tertiary PPI to be measured. Optionally, images canbe constructed for visualization showing each signal with overlay (e.g.,red and green overlay).

In the various kits of the invention, each kit can include instructionsor packaging materials that describe how to use a compound orcomposition (e.g., a reagent such as a primary antibody, a secondaryantibody, a labeled oligonucleotide probe that hybridizes with theamplification product) of the kit. Within the kit, the secondaryantibody may be uncojugated or conjugated to an oligonucleotide (makingthe secondary antibody a proximity probe). The kits may also comprise,e.g., polymerase (for the amplification reaction), ligase (for theligation reaction), a buffering agent, a preservative, or a proteinstabilizing agent.

The kits may also comprise components necessary for detecting the label(e.g., an enzyme or substrate). The kit may also contain a controlsample or a series of control samples that can be assayed and comparedto a test sample. Each kit can include one or more containers forindividually enclosing each component of the kit. Containers of the kitscan be of any suitable material, e.g., glass, plastic, metal, etc., andof any suitable size, shape, or configuration. The one or morecontainers are can be enclosed within outer packaging.

The kits of the invention may be used by contacting the cell-containingsample with the primary antibodies (allowing the primary antibodies tobind to their respective protein targets), contacting the sample withthe proximity probes (allowing the proximity probes to bind to theirrespective antibody targets), allowing the nucleic acid strands (alsoreferred to as aims) of the proximity probes to ligate if binding inclose proximity (adding ligase if necessary), amplifying the ligationproduct (adding polymerase if necessary) to produce an amplificationproduct, and measuring the amplification product corresponding to theinteraction of each pair of protein targets within the PPI. Measurementof the amplification product can be made using methods known in the artfor detecting and quantifying nucleic acid amplification products, e.g.,by adding a labeled oligonucleotide that hybridizes to a sequence of theamplification product, and analyzing the sample by visualizing theamplification product (as represented by the label signal) with anappropriate visualization device, such as a confocal or fluorescencemicroscope.

Another aspect of the invention concerns a method for measuringprotein-protein interactions having three or more binding partners (atertiary interaction) in a sample, comprising:

contacting the sample with three or more primary antibodies to three ormore corresponding target binding partners within a target tertiaryinteraction;

contacting the sample with three or more proximity probes (first,second, and third proximity probes) comprising a secondary antibody thatbinds to the corresponding first antibody, wherein each proximity probehas an oligonucleotide conjugated thereto;

-   -   wherein when the oligonucleotides of the first and second        proximity probes are in sufficient proximity to each other, the        oligonucleotides of the first and second proximity probes        interact in the presence circle-forming oligonucleotides by        enzymatic ligation and form a circular product that is amplified        by rolling circle replication, producing a first amplification        product; and    -   wherein when the oligonucleotides of the second and third        proximity probes are in sufficient proximity to each other, the        oligonucleotides of the second and third proximity probes        interact in the presence of circle-forming oligonucleotides by        enzymatic ligation and form a circular product that is amplified        by rolling circle replication, producing a second amplification        product; and measuring the first and second amplification        products.

In some embodiments, measurement of the first and second amplificationproducts comprises contacting the sample with two or more labeledoligonucleotides, comprising a first labeled oligonucleotide thathybridizes to a sequence of the first amplification product, and asecond labeled oligonucleotide that hybridizes to a sequence of thesecond amplification product, to produce labeled amplification products,wherein the labels are distinguishable from one another; and measuringthe PPIs by visualizing the labeled amplification products. Anadditional primary antibody, proximity probe, and labeledoligonucleotide can be used for each additional target binding partnerwithin the tertiary PPI.

Another aspect of the invention concerns a method for the identificationof a biomarker, comprising selecting two or more target binding partnerswithin a cancer sample; generating a PPI profile for the two or moretarget binding partners; and comparing the PPI profile to theresponsiveness of the cancer to a treatment in vitro and/or in vivo (forexample, in xenograft animal models or human subjects). Correlationbetween the PPI profile to the responsiveness of the cancer to atreatment of the cancer in vitro and/or in vivo is indicative of abiomarker for treatment responsiveness for the cancer. In someembodiments, the treatment is a kinase inhibitor (e.g., a tyrosinekinase inhibitor (TKI)). In some embodiments, at least one of the two ormore target binding partners comprises a receptor tyrosine kinase (RTK).In some embodiments, RTK is within RTK class I-XVII. Preferably,responsiveness of the treatment to the cancer in vitro, or in vivo withxenograft animal models, is determined with the subject's cancer cells;however, cancer cell lines known to be predictive of responsiveness tothe subject's cancer type may be utilized to compare with the PPIprofile.

Another aspect of the invention concerns a method for identifying anagent as a PPI modulator, comprising: contacting cancer cells with acandidate agent in vitro or in vivo; and determining whether thecandidate agent modulates a selected PPI in a sample of the cancercells. The candidate agent may be any substance that potentiallymodulates (increases, decreases, or otherwise alters) the PPI. Thecandidate agent may be a small molecule, polypeptide, or nucleic acid,for example. Determination of changes in PPI can be made by comparing afirst PPI profile of the cancer cells obtained prior to the contactingstep to a second PPI profile of the cancer cells obtained after thecontacting step, wherein a change in the PPI is indicative of a PPImodulator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows physical mapping of protein-protein interactions involvingEGFR, showing 201 interactions with EGFR. FIG. 1 demonstrates howinteraction data (e.g., from literature or affinity-purification massspectrometry) can be used to generate pairs of proteins to study asbiomakers for treatment efficacy using proximity ligation assays.

FIGS. 2A and 2B are diagrams showing the proximity ligation assay (PLA)scheme.

FIGS. 3A-3F show results of an EGFR-GRB2 PLA in situ. Red dots aresignal of EGFR-GRb2 interaction detected by PLA using confocal imageswith 10 z-sections merged.

FIG. 3A shows strong signals in PC9 cell line that are dramaticallyreduced after treated with erlotinib (FIG. 3B). Negative controls areshown in FIGS. 3C to 3F: no anti-rabbit 2^(nd) antibody with PLUS DNAchain (FIG. 3C); no anti-mouse 2^(nd) antibody with MINUS DNA chain(FIG. 3D), no rabbit anti-Grb2 antibody (FIG. 3E); no mouse anti-EGFRantibody (FIG. 3F).

FIGS. 4A and 4B show results of EGFR-Grb2 PLA in the PC9 lung cancercell line. PC9 lung cancer cells harboring an activating EGFR mutationwere examined for PLA using EGFR and Grb2 antibodies. Control untreatedcells (FIG. 4A) were examined as well as cells exposed to erlotinib, asmall molecule epidermal growth factor tyrosine kinase inhibitor (EGFRTKI) (FIG. 4B). Erlotinib inhibits EGFR tyrosine phosphorylation andprevents Grb2 binding (data now shown) thus serving as a perturbation toexamine PLA. As shown here, the EGFR-Grb2 PLA signal (red dots) isstrong across multiple cells and strongly repressed by erlotinib. Bluestain=DAPI that stains nuclei. This figure also demonstrates thefeasibility of performing EGFR-Grb2 PLA (and potential other useful PLAbiomarkers) on low numbers of cells (and may work with cytologyspecimens or other scant tumor biopsy specimens).

FIGS. 5A and 5B show results of EGFR-Grb2 PLA in lung cancer tumortissues (in vivo). Formalin-fixed paraffin-embedded (FFPE) tumor tissuefrom a surgically resected adenocarcinoma of the lung was used forEGFR-Grb2 PLA. As a negative control, FFPE spleen tissue was chosen, asthis tissue does not express EGFR protein. As shown in FIG. 5A, the lungadenocarcinoma has strong signal (red dots), compared to spleen that hasno signal, shown in FIG. 5B. The blue signal is DAPI that stains fornuclei.

FIGS. 6A and 6B show results of ALK-Grb2 PLA in H3122 cells. PLAs havealso been generated for detecting interactions between ALK and Grb2.H3122 cells harboring an ELM4-ALK gene fusion were examined usingprimary antibodies against the ALK receptor tyrosine kinase and Grb2.The negative control consisted of the same assay but leaving out theGrb2 antibody. In FIG. 6A, the small red foci are identified, indicativeof ALK-Grb2 interactions, while no foci are observed in the negativecontro (FIG. 6B). This indicates the PLAs can be generated to identifytumors driven by other receptor tyrosine kinases (beyond EGFR),including EML4-ALK transformed lung cancers.

FIG. 7 shows results of examination of lung cancer cell lines and spleentissue for EGFR:Grb2 PLA signals related to EGFR mutation status anddrug sensitivity. This included cells harboring EGFR mutation (HCC827,PC9, H4006, H1650). All these cells had high levels of EGFR:Grb2 PLAsignal. H1648, H322, H358, and H292 cells also demonstrated intermediateto higher levels of PLA signal; these cells have wildtype EGFR yet havesome degree of sensitivity to EGFR TKI (erlotinib). Finally, A549, H23,and H460 cells are resistant to EGFR TKI (erlotinib) and have theweakest signals amongst the lung cancer cell lines studied. These dataindicate that not only does EGFR:Grb2 PLA signal correspond to presenceof EGFR mutation status but it also may correlate with or predictsensitivity to EGFR TKI.

FIG. 8 shows results of PLA examination of a tissue microarray (TMA)that was produced from 21 FFPE lung cancer tumors, 14 without EGFRmutations and 7 with activating EGFR mutations. Included in this arraywas normal spleen tissue as a negative control. Tissues were examinedusing EGFR:Grb2 PLA. Of the 7 tumors with EGFR mutation, 6 had thestrongest signals across the sample set. One tumor with EGFR mutationthat lacked EGFR:Grb2 signal also has a corresponding PIK3CA mutationleading to enhanced PIK3CA activity. The 14 cases without EGFR mutationhad lower degrees of signal compared to the 6 positive cases with EGFRmutation. Assuming a signalnoise cutoff of 10 (noise level=zerobackground of spleen tissue), 6 of the 14 cases had positive levels ofEGFR:Grb2 PLA signal. This indicates a group of tumors lacking EGFRmutation that nonetheless may have EGFR pathway activation.

FIGS. 9A and 9B show results of PLA assays revealing that EGFR-GRB2interactions are independent of expression and generally correlated withEGFR phosphorylation. PLA assays were performed across 9 non-small celllung cancer (NSCLC) cell lines (PC9, H1650, HCC827, H4006, H1299, H1648,H322, A549, and H23) and analyzed by confocal microscopy at 400×magnification (FIG. 9A). Images represent maximum projections of 12×0.76μm z-slices. Foci are imaged in the far-red spectrum (Cy5) and nucleiwere imaged in the UV region with DAPI. Foci were detected in all EGFRmutant cell lines, while several cell lines (H1299 and H23) have nearlyundetectable levels foci and serve as biological negative controlsdemonstrating the specificity of PLA. Intermediate levels of foci weredetected in H322 and H1648, both of which exhibit some degree oferlotinib sensitivity. These results show that EGFR-GRB2 foci correlatewith drug sensitivity. Immunoblot analysis of the 9 NSCLC cell lines (50μg each) showed that all cells expressed detectable levels of both EGFRand GRB2, while phosphorylation of tyrosine 1068 of EGFR was detectedonly in EGFR mutant cell lines. These results demonstrate that thepresence of the EGFR and GRB2 is necessary but not sufficient forinteraction. The detection of signal (visible foci) in drug sensitivityfurther supports the use of PPI as a biomarker of drug sensitivity.

FIG. 10 shows PLA results revealing that EGFR-GRB2 interactionscorrelate with mutational status and are abrogated with TKI treatment invivo. Patient-derived xenografts were grown in nude mice and treatedwith erlotinib for 28 days or with vehicle control. Slides were preparedfrom excised tumor tissue (verified by pathologist) and analyzed by PLAusing 200× magnification. Foci are imaged in the far-red spectrum (Cy5)and nuclei were detected in the UV region with DAPI. Tumors derived frompatients with an activating EGFR mutation had very high levels ofEGFR-GRB2 detectable by PLA, while EGFR wildtype tumors had markedlylower levels. Importantly, PLA signal in the EGFR mutant tumors wasconsiderably reduced after administration of erlotinib. These resultsindicate that mutation status is correlated with EGFR-GRB2 foci in vivoand that foci are correlated with erlotinib activity.

FIG. 11 shows PLA revealing that normal tissues have low to undetectableEGFR-GRB2 interactions. Eight normal tissues were assayed by PLA forEGFR-GRB2 and imaged concordantly with staining for theepithelial-origin marker cytokeratin using (Cy3). As expected, lowlevels of foci are found in the trophoblasts in placenta and epithelialcells of the gall bladder. Foci were not detected in pancreas, spleen,colon, heart and normal lung. Thus, EGFR-GRB2 interactions aredetectable where EGFR is naturally expressed, but at low levelsconsistent with the growth-arrested state of most normal tissues.

FIGS. 12A-1-12D-1 show PLA results revealing EGFR-GRB2 interactions inlung tumor tissues and co-localization with cytokeratin. Tissues wereassayed by PLA in tumor microarray format and analyzed as in FIG. 11.Foci are readily detectable in patient 2 (FIGS. 12B-1 and 12B-2) andpatient 4 (FIGS. 12D-1 and 12D-2), and absent in patient 1 (FIGS. 12A-1and 12A-2) and patient 3 (FIGS. 12C-1 and 12C-2). Importantly, the fociobserved in patients 2 and 4 are only found in cytokeratin-positiveregions of the specimen, indicating the specificity of PLA in tissues.For each patient, the left panels (FIGS. 12A-1, 12B-1, 12C-1, and 12D-1)show nuclei as blue (DAPI), cytokeratin as green (Cy3) and PLA foci asred (Cy5). In each patient, the right panels (FIGS. 12A-2, 12B-2, 12C-2,and 12D-2) are the same images with the green channel removed forclarity. These results demonstrate that PLA signals are specific forepithelial-derived tissue and provide a tool to quantify signal onlywithin the tumor.

FIGS. 13A and 13B show EGFR-GRB2 PLA demonstrating that PPI areindependent of expression levels in lung tumor tissue. PLA wereperformed and imaged as above on two patients, one harboring anactivating mutation in EGFR (FIG. 13A) and the other with wildtype EGFR(FIG. 13B). Both patients express EGFR protein shown on the left, whereCy5-labelled secondary antibodies were used to image EGFR expression byimmunofluorescence. Although patient B has higher levels of EGFR proteinexpression (FIG. 13B), the PLA signal is higher for patient A (FIG.13A). These data suggest that PLA signal may be a powerful indicator ofEGFR signaling activity.

FIGS. 14A and 14B show PLA results demonstrating that PLA foci can bevisualized with immuno-fluorescence or brightfield microscopy. PLA wereperformed on sequential tissue sections and processed at the same time.FIG. 14A was processed using brightfield detection reagents (horseradishperoxidase-labeled oligonucleotides) and counter-stained withhematoxylin. FIG. 14B was processed using immunofluorescence detectionreagents (Cy5-labeled oligonucleotides) and nuclei stained with DAPI andtissue autofluorescence imaged on the FITC channel. Images were obtainedat 400× magnification on brightfield or immunofluorescent platforms,respectively. Similar results were obtained with both methods ofdetection, indicating that PLA can be performed using only lightmicroscopy.

FIGS. 15A-15C show PLA results demonstrating that PLA can be used tomonitor phosphorylation status. PLA were performed in three cell linesto assess phosphorylation dynamics as phosphorylation of RTKs isrequired for binding of downstream effectors. A549 cells were stimulatedwith 50 ng/ml EGF for various timepoints and phosphorylation of EGFR wasmonitored by PLA using a single antibody to phosphor-EGFR_(Y1068) (FIG.15A). Unstimulated A549 cells have low levels of EGFR phosphorylation,which quickly increases before diminishing. PC9 cells were treated for 3hours with erlotinib and assayed as above (FIG. 15B). 3 hours oferlotinib treatment abolishes the phosphorylation of EGFR. For FIGS. 15Aand 15B, immunoblots confirmed the changes in phosphorylation status(FIG. 15A-1 and 15B-1, respectively). H3122 cells were assayed by PLAusing an antibody to ALK and total phosphotyrosine to monitor the changein phosphorylation of ALK in response to crizotinib (FIG. 15C). 3 hoursof crizotinib treatment abolishes the phosphorylation of ALK.Importantly, these assays demonstrate that phosphorylation dynamics canbe monitored by PLA and that using an RTK antibody paired with a “pan”phosphotyrosine is a viable approach.

FIGS. 16A-16D show results of ALK-Shc1 PLA in EML4-ALK rearranged lungcancer cells. FIGS. 16B and 16D show interaction foci between ALK andShc1 in EML4-ALK rearranged lung cancer cells that disappear with ALKTKI (crizotinib therapy). FIGS. 16A and 16C show pTyr ALK.

FIG. 17 shows PLA results demonstrating that PLA facilitate theobservation of pharmacodynamic effects of kinase inhibitors on signalingnetworks. Tumor xenografts were established in mice with either H1650(EGFR mutant) or H322 (EGFR wildtype), treated with or without erlotiniband allowed to grow for 18 days. Formalin-fixed, paraffin-embedded tumortissue was analyzed by PLA for the interaction of EGFR-GRB2 (left) andEGFR-SHC1 (right). Nuclei are shown in blue, PLA signal is red andcytokeratin is green. For clarity, identical images are shown with andwithout cytokeratin staining for each tumor. H1650 cells exhibit highlevels of EGFR-GRB2 and considerably higher levels of EGFR-Shc1,consistent with the presence of an activating EGFR mutation. H322 cellshave lower levels of EGFR interactions, but still show a reduction uponTKI treatment. These data indicate that PLA signal is markedly reducedupon TKI treatment, specifically within the tumor tissues and can beused to evaluate target-specific drug efficacy in vivo.

DETAILED DISCLOSURE OF THE INVENTION

Proximity ligation assay (PLA)-based biomarkers could be an importantfuture direction in personalized medicine. They could be useful forcancer prognosis as well as decision making regarding targeted therapy.The present invention includes assays for protein-protein interactions(PPI) that can be used to determine, for example, when an activatedreceptor tyrosine protein kinase binds a signal transduction moleculesuch as GRB2. This assay uses PLAs to quantitatively measure PPI intissue and cell samples such as that prepared for microscopy. The PLAtypically involves primary and secondary antibodies binding two separateproteins in a sample; short nucleic acid strands are attached to thesecondary antibodies which are ligated into circles and then amplifiedand labeled, e.g., with fluorescent probes. The inventors haveestablished in cell lines that EGFR-GRB2 PPIs distinguish populations oflung cancer cells, are independent of EGFR expression level andgenerally correlate with EGFR phosphorylation. The assay has also beenshown to work in FFPE patient tissue samples. The assay can be used todetermine if a patient's receptor tyrosine kinase pathway is activatedand, therefore, may be a good candidate for intervention with a tyrosinekinase inhibitor.

The assays of the invention represent a platform technology expandableto a larger spectrum of tyrosine kinases and possible signaltransduction docking proteins. The assays may be used as a surrogate fortyrosine phosphorylation status, as tyrosine phosphorylation is noteasily discerned in FFPE slides without the slides being prepared in aspecial manner. The assays of the invention can read out pathwayactivation resulting from mutation, gene amplification, or autocrinefactors. Moreover, not all tyrosine kinases have phosphorylationspecific antibodies, and it may be easier to make antibodies to theoverall tyrosine kinase protein and its adaptor proteins. Given themarket of tyrosine kinase inhibitors, both approved and in development,this technology can provide a number of potential biomarker strategiesincluding diagnostics for drug response or companion diagnostics.

One aspect of the invention concerns a method for assessing thesensitivity of a malignancy to a treatment based on PPI. The method forassessing the sensitivity of a malignancy to a treatment comprisescomparing a protein-protein interaction (PPI) profile obtained from asample of the malignancy to a reference PPI profile. The sample PPIprofile represents the abundance of target binding partners that are inproximity to each other within the sample. A negative result (lack ofPPI) in a sample would indicate a lack of sensitivity to the PPImodulator and be useful clinically to avoid giving patients unnecessaryPPI modulator therapy. A positive result (presence of PPI) wouldindicate that the malignancy is potentially sensitive to the PPImodulator and allow the clinician to give the PPI modulator therapy tothose patients who would be most likely to benefit. As a specificexample, the method may be used for determining whether there is no EGFsignaling in a sample such as spleen tissue, and this kind of a negativeresult can be useful clinically to avoid giving patients unnecessary EGFreceptor inhibitor therapy, or alternatively be used to give the therapyto those patients who would be most likely to benefit.

PPI profiles (e.g., sample PPI profile, and reference PPI profile) mayeach be expressed as a value representative of the abundance of targetbinding partners in proximity to each other within the sample. Thesample PPI profile and reference PPI profile may be expressed by anymethod useful for comparison purposes, such as a numeric value, score,cutoff (threshold), or other expression. For example, a negative resultin which a PPI profile in a sample does not reach the cutoff would beuseful clinically to avoid giving patients unnecessary PPI modulatortherapy. A positive result in which a PPI profile in a sample is at orabove the cutoff would indicate potential sensitivity and allow theclinician to give the PPI modulator therapy to those patients who wouldbe most likely to benefit.

In some embodiments of the methods and kits of the invention, the targetPPI is that of a known cancer signaling network. Binding members of atarget PPI may include protein products of oncogenes or tumor suppressorgenes, for example. In some embodiments, the sample PPI profilerepresents the abundance of target binding partners that are inproximity to each other within the sample, wherein at least one of thetarget binding partners is selected from among EGFR, ALK, MET, IGFR,Src, ErbB3, Mig6, Grb2, Sts1, p85, and Hsp90.

In some embodiments of the methods and kits of the invention, the targetbinding partners are selected from among EGFR and ErbB3; EGFR and Grb2;EGFR and Mig6; EGFR and Sts1, EGFR and Src; EGFR and Hsp90; ErbB3 andp85; ALK and EML4; MET and Gab1; IGFR and IRS; Hsp90 and Cdc37; ALK andShc1; and EGFR and Shc1 .

In some embodiments of the methods and kits of the invention, the PPIcomprises a tyrosine kinase, such as the human tyrosine kinases listedin Tables 3 and 4. In some embodiments, the PPI comprises an interactionbetween the binding partners listed in Table 5 (human tyrosine kinaseinteractions).

PLAs can be used to generate biomarkers against receptor tyrosinekinases (RTKs). RTKs are important proteins in cancer and highly“druggable” targets. Approximately twenty different RTK classes havebeen identified thus far. In some embodiments of the methods and kits ofthe invention, at least one of the target binding partners is an RTK. Insome embodiments of the methods and kits of the invention, at least oneof the target binding partners is of an RTK class selected from amongRTK class I (EGF receptor family; ErbB family), RTK class II (insulinreceptor family), RTK class III (PDGF receptor family), RTK class IV(FGF receptor family) RTK class V (VEGF receptors family), RTK class VI(HGF receptor family), RTK class VII (Trk receptor family), RTK classVIII (Eph receptor family), RTK class IX (AXL receptor family), RTKclass X (LTK receptor family), RTK class XI (TIE receptor family), RTKclass XII (ROR receptor family), XIII (DDR receptor family), RTK classXIV (RET receptor family), RTK class XV (KLG receptor family), RTK classXVI (RYK receptor family), and RTK class XVII (MuSK receptor family).

In some embodiments of the methods and kits of the invention, one ormore of the binding partners of the target PPI have one or more sequencemutations that are known to be associated with occurrence of themalignancy. In other embodiments, the binding partners of the target PPIdo not harbor (lack) any sequence mutations known to be associated withoccurrence of the malignancy, or with sensitivity to a treatment such asa PPI modulator. In some embodiments, the binding partners of the targetPPI and the downstream effectors of the target PPI do not harbor anysequence mutations known to be associated with occurrence of themalignancy, or with sensitivity to a treatment such as a PPI modulator.Such sequence aberrations in a subject or in a sample can be detectedusing methods known in the art (e.g., mutation analysis). Typically,abnormalities in nucleic acid sequences are identified by comparison toreference sequence data (sequences of normal cells or cancer cells) ondatabases, such as GenBank and EMBL, and specific data resources such asCancer Gene Census (mutated genes causally implicated in human cancer),COSMIC (Catalogue of Somatic Mutations in Cancer), and CGP ResequencingStudies (somatic mutations from large scale resequencing of genes inhuman cancer). Mutations causing or contributing to cancer may belarge-scale mutations, involving the deletion or addition of a portionof a chromosome, or small-scale mutations, including point mutations,deletions, insertions, which may occur in the promoter region of a geneand affect its expression, may occur in the coding sequence and alterthe stability or function of the gene's protein product.

The sample may be any cell sample potentially harboring the targetprotein(s). For example, a cytology sample may be obtained from a tissueselected from breast, ovaries, esophagus, stomach, colon, rectum, anus,bile duct, brain, endometrium, lung, liver, skin, prostate, kidney,nasopharynx, pancreas, head and neck, kidney, lymphoma, leukemia,cervix, and bladder. The sample may be a solid or non-solid tumorspecimen. The tumor specimen may be a carcinoma. The sample may be a newcancer, recurrent cancer, primary cancer, or metastasized (secondary)cancer.

The sample may be obtained by methods known in the art, such as surgery,biopsy, or from blood (e.g., circulating tumor cells), ascites, orpleural effusion. The sample may be processed using methods known in theart. For example, the sample may be fresh, frozen, or formalin-fixed andparaffin-embedded (FFPE).

Preferably, the treatment against which the sample is being assessed forsensistivity/resistance is a PPI modulator (i.e., a PPI inhibitor or PPIinducer). However, the treatment may be a treatment other than a PPImodulator.

Another aspect of the invention concerns a method for treating amalignancy in a subject, comprising administering a protein-proteininteraction (PPI) modulator to the subject, wherein the subject ispredetermined to be sensitive to the PPI modulator based on a PPIprofile obtained from a sample of the malignancy. In some embodiments,the PPI modulator is an inhibitor of the PPI of the PPI profile (i.e., aPPI inhibitor). In some embodiments, the PPI modulator is an inducer ofthe PPI of the PPI profile (i.e., a PPI inducer).

Another aspect of the invention concerns a method for treating amalignancy in a subject, comprising:

(a) assessing the sensitivity of a malignancy in the subject, comprisingcomparing a protein-protein interaction (PPI) profile obtained from asample of the malignancy to a reference PPI profile; and

(b) administering a PPI modulator to the subject if the malignancy isassessed to be sensitive to the PPI modulator; and withholding the PPImodulator from the subject if the malignancy is assessed to be resistantto the PPI modulator. In some embodiments, the PPI modulator is aninhibitor of the PPI of the PPI profile (i.e., a PPI inhibitor). In someembodiments, the PPI modulator is an inducer of the PPI of the PPIprofile (i.e., a PPI inducer).

The invention includes also includes kits useful for carrying outmethods of the invention, (e.g., methods for the classification ofcancers as sensitive or resistant to treatments based on protein-proteininteractions, treatment of cancer, identification of biomarkers,identification of protein-protein interaction modulators, and selectionof cancer treatments). Thus, one aspect of the invention concerns a kitfor detecting the proximity of target binding partners within a cancerprotein-protein interaction network, comprising a primary antibody to atleast one of the target binding partners, and a proximity probecomprising a secondary antibody (that binds to the primary antibody)with an oligonucleotide conjugated thereto. In preferred embodiments,the kit comprises:

a first primary antibody to a first target binding partner;

a second primary antibody to a second target binding partner;

a first proximity probe comprising a first secondary antibody (thatbinds to the first primary antibody) with an oligonucleotide conjugatedthereto; and

a second proximity probe comprising a second secondary antibody (thatbinds to the second primary antibody) with an oligonucleotide conjugatedthereto, wherein when the oligonucleotides of the first and secondproximity probes are in sufficient proximity to each other, theoligonucleotides of the proximity probes interact in the presencecircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing an amplification product. Optionally, the kit furthercomprises a labeled oligonucleotide probe that hybridizes with theamplification product, allowing detection and quantification of theamplification product (representing the association (close proximity) ofthe target binding partners).

Another aspect of the invention concerns a kit for detecting theproximity of target binding partners within tertiary PPI (a complexhaving three or more protein binding partners), referred to herein asthe “tertiary interaction kit”. The target PPI may be within a cancersignaling network, but is not limited to such applications. The tertiaryinteraction kit can be used to study viruses, for example, in which aviral protein forms an interaction with protein A and protein B in acell, giving rise to a tertiary complex. Identification of this tertiaryPPI may be diagnostic of an active infection or yield importantinformation about the prognosis or predict the correct therapy. Incancer, a tertiary complex may provide important information about thetherapeutic efficacy.

The tertiary interaction kit comprises:

a first primary antibody to a first target binding partner of thetertiary interaction;

a second primary antibody to a second target binding partner of thetertiary interaction;

a third primary antibody to a third target binding partner of thetertiary interaction;

a first proximity probe comprising a first secondary antibody (thatbinds to the first primary antibody) with an oligonucleotide conjugatedthereto; and

a second proximity probe comprising a second secondary antibody (thatbinds to the second primary antibody) with an oligonucleotide conjugatedthereto;

a third proximity probe comprising a third secondary antibody (thatbinds to the third primary antibody) with an oligonucleotide conjugatedthereto;

wherein when the oligonucleotides of the first and second proximityprobes are in sufficient proximity to each other, the oligonucleotidesof the first and second proximity probes interact in the presencecircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing a first amplification product; and

wherein when the oligonucleotides of the second and third proximityprobes are in sufficient proximity to each other, the oligonucleotidesof the second and third proximity probes interact in the presence ofcircle-forming oligonucleotides by enzymatic ligation and form acircular product that is amplified by rolling circle replication,producing a second amplification product. Optionally, the kit detectingproximity of binding partners within a tertiary PPI further comprises afirst labeled oligonucleotide probe that hybridizes with the firstamplification product, allowing detection and quantification of thefirst amplification product (representing the association (closeproximity) of the first and second target binding partners), and asecond labeled oligonucleotide probe that hybridizes with the secondamplification product, allowing detection and quantification of thesecond amplification product (representing the association of the secondand third target binding partners). Each primary antibody is of adifferent species (e.g., mouse, rabbit, and goat, for example).Preferably, the label of the first labeled oligonucleotide yields asignal (e.g., a color) that is distinguishable from that of the secondlabeled oligonucleotide (red and green, for example). An additionalprimary antibody, proximity probe, and (optionally) oligonucleotideprobe can be included in the kit for each additional target bindingpartner within the tertiary PPI to be measured. Optionally, images canbe constructed for visualization showing each signal with overlay (e.g.,red and green overlay).

In the various kits of the invention, each kit can include instructionsor packaging materials that describe how to use a compound orcomposition (e.g., a reagent such as a primary antibody, a secondaryantibody, a labeled oligonucleotide probe that hybridizes with theamplification product) of the kit. Within the kit, the secondaryantibody may be uncojugated or conjugated to an oligonucleotide (makingthe secondary antibody a proximity probe). The kits may also comprise,e.g., polymerase (for the amplification reaction), ligase (for theligation reaction), a buffering agent, a preservative, or a proteinstabilizing agent. The kits may also comprise components necessary fordetecting the label (e.g., an enzyme or substrate). The kit may alsocontain a control sample or a series of control samples that can beassayed and compared to a test sample. Each kit can include one or morecontainers for individually enclosing each component of the kit.Containers of the kits can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.The one or more containers are can be enclosed within outer packaging.

The kits of the invention may be used by contacting the cell-containingsample with the primary antibodies (allowing the primary antibodies tobind to their respective protein targets), contacting the sample withthe proximity probes (allowing the proximity probes to bind to theirrespective antibody targets), allowing the nucleic acid strands (alsoreferred to as aims) of the proximity probes to ligate if binding inclose proximity (adding ligase if necessary), amplifying the ligationproduct (adding polymerase if necessary) to produce an amplificationproduct, and measuring the amplification product corresponding to theinteraction of each pair of protein targets within the PPI. Measurementof the amplification product can be made using methods known in the artfor detecting and quantifying nucleic acid amplification products, e.g.,by adding a labeled oligonucleotide that hybridizes to a sequence of theamplification product, and analyzing the sample by visualizing theamplification product (as represented by the label signal) with anappropriate visualization device, such as a confocal or fluorescencemicroscope.

Another aspect of the invention concerns a method for measuringprotein-protein interactions having three or more binding partners (atertiary interaction) in a sample, comprising:

contacting the sample with three or more primary antibodies to three ormore corresponding target binding partners within a target tertiaryinteraction;

contacting the sample with three or more proximity probes (first,second, and third proximity probes) comprising a secondary antibody thatbinds to the corresponding first antibody, wherein each proximity probehas an oligonucleotide conjugated thereto;

-   -   wherein when the oligonucleotides of the first and second        proximity probes are in sufficient proximity to each other, the        oligonucleotides of the first and second proximity probes        interact in the presence circle-forming oligonucleotides by        enzymatic ligation and form a circular product that is amplified        by rolling circle replication, producing a first amplification        product; and    -   wherein when the oligonucleotides of the second and third        proximity probes are in sufficient proximity to each other, the        oligonucleotides of the second and third proximity probes        interact in the presence of circle-forming oligonucleotides by        enzymatic ligation and form a circular product that is amplified        by rolling circle replication, producing a second amplification        product; and

measuring the first and second amplification products.

In some embodiments, measurement of the first and second amplificationproducts comprises contacting the sample with two or more labeledoligonucleotides, comprising a first labeled oligonucleotide thathybridizes to a sequence of the first amplification product, and asecond labeled oligonucleotide that hybridizes to a sequence of thesecond amplification product, to produce labeled amplification products,wherein the labels are distinguishable from one another; and measuringthe PPIs by visualizing the labeled amplification products. Anadditional primary antibody, proximity probe, and labeledoligonucleotide can be used for each additional target binding partnerwithin the tertiary PPI.

Another aspect of the invention concerns a method for the identificationof a biomarker, comprising selecting two or more target binding partnerswithin a cancer sample; generating a PPI profile for the two or moretarget binding partners; and comparing the PPI profile to theresponsiveness of the cancer to a treatment in vitro and/or in vivo (forexample, in xenograft animal models or human subjects). Correlationbetween the PPI profile to the responsiveness of the cancer to atreatment of the cancer in vitro and/or in vivo is indicative of abiomarker for treatment responsiveness for the cancer. In someembodiments, the treatment is a kinase inhibitor (e.g., a tyrosinekinase inhibitor (TKI)). In some embodiments, at least one of the two ormore target binding partners comprises a receptor tyrosine kinase (RTK).In some embodiments, RTK is within RTK class I-XVII. Preferably,responsiveness of the treatment to the cancer in vitro, or in vivo withxenograft animal models, is determined with the subject's cancer cells;however, cancer cell lines known to be predictive of responsiveness tothe subject's cancer type may be utilized to compare with the PPIprofile.

Another aspect of the invention concerns a method for identifying anagent as a PPI modulator, comprising: contacting cancer cells with acandidate agent in vitro or in vivo; and determining whether thecandidate agent modulates a selected PPI in a sample of the cancercells. The candidate agent may be any substance that potentiallymodulates (increases, decreases, or otherwise alters) the PPI. Thecandidate agent may be a small molecule, polypeptide, or nucleic acid,for example. Determination of changes in PPI can be made by comparing afirst PPI profile of the cancer cells obtained prior to the contactingstep to a second PPI profile of the cancer cells obtained after thecontacting step, wherein a change in the PPI is indicative of a PPImodulator.

By inhibiting the growth of cells proliferating in an aberrant manner,the methods, PPI modulators, and compositions of the present inventioncan be used to treat a number of cell proliferation disorders, such ascancers, including, but not limited to, leukemias and lymphomas, such asacute lymphocytic leukemia, acute non-lymphocytic leukemias, chroniclymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's Disease,non-Hodgkin's lymphomas, and multiple myeloma, childhood solid tumorssuch as brain tumors, neuroblastoma, retinoblastoma, Wilms' Tumor, bonetumors, and soft-tissue sarcomas, common solid tumors of adults such aslung cancer, colon and rectum cancer, breast cancer, prostate cancer,urinary cancers, uterine cancers, bladder cancers, oral cancers,pancreatic cancer, melanoma and other skin cancers, stomach cancer,ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroidcancer, esophageal cancer, and testicular cancer. The methods of thesubject invention can be carried out in vivo or in vitro, to inhibit thegrowth of cells (e.g., cancer cells) in humans and non-human mammals.

In some embodiments, the proliferation disorder to be treated is acancer producing a tumor characterized by aberrant protein-proteininteraction.

The methods of the present invention can be advantageously combined withat least one additional treatment method, including but not limited to,surgery, chemotherapy, radiation therapy, or any other therapy known tothose of skill in the art for the treatment and management ofproliferation disorders such as cancer.

While PPI modulators can be administered to cells in vitro and in vivoas isolated agents, it is preferred to administer PPI modulators as partof a pharmaceutical composition, in association with at least onepharmaceutically acceptable carrier. The pharmaceutical composition canbe adapted for various routes of administration, such as enteral,parenteral, intravenous, intramuscular, topical, subcutaneous, and soforth. Administration can be continuous or at distinct intervals, as canbe determined by a person of ordinary skill in the art.

The PPI modulators utilized in the invention can be formulated accordingto known methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin, E. W., 1995, Easton Pa.,Mack Publishing Company, 9^(th) ed.) describes formulations which can beused in connection with the subject invention. Formulations suitable foradministration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids that form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts of compounds may be obtained usingstandard procedures well known in the art, for example, by reacting asufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for examplecalcium) salts of carboxylic acids can also be made.

As used herein, the term “analogs” refers to compounds which aresubstantially the same as another compound but which may have beenmodified by, for example, adding side groups, oxidation or reduction ofthe parent structure. Analogs of particular PPIs, and other agentsdisclosed herein, can be readily prepared using commonly known standardreactions. These standard reactions include, but are not limited to,hydrogenation, alkylation, acetylation, and acidification reactions.Chemical modifications can be accomplished by those skilled in the artby protecting all functional groups present in the molecule anddeprotecting them after carrying out the desired reactions usingstandard procedures known in the scientific literature (Greene, T. W.and Wuts, P.G.M. “Protective Groups in Organic Synthesis” John Wiley &Sons, Inc. New York. 3rd Ed. pg. 819, 1999; Honda, T. et al. Bioorg.Med. Chem. Lett., 1997, 7:1623-1628; Honda, T. et al. Bioorg. Med. Chem.Lett., 1998, 8:2711-2714; Konoike, T. et al. J. Org. Chem., 1997,62:960-966; Honda, T. et al. J. Med. Chem., 2000, 43:4233-4246; each ofwhich are hereby incorporated herein by reference in their entirety).Analogs, fragments, and variants of PPI modulators exhibiting thedesired biological activity (such as induction of cell death,cytotoxicity, cytostaticity, induction of cell cycle arrest, etc.) canbe identified or confirmed using cellular assays or other in vitro or invivo assays.

Therapeutic application of the PPI modulators and compositionscomprising them can be accomplished by any suitable therapeutic methodand technique presently or prospectively known to those skilled in theart.

Active agents such as PPI modulators may be locally administered at oneor more anatomical sites, such as sites of unwanted cell growth (such asa tumor site, e.g., injected or topically applied to the tumor),optionally in combination with a pharmaceutically acceptable carriersuch as an inert diluent. PPI modulators may be systemicallyadministered, such as intravenously or orally, optionally in combinationwith a pharmaceutically acceptable canier such as an inert diluent, oran assimilable edible carrier for oral delivery. They may be enclosed inhard or soft shell gelatin capsules, may be compressed into tablets, ormay be incorporated directly with the food of the patient's diet. Fororal therapeutic administration, the PPI modulators may be combined withone or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the PPI modulators maybe incorporated into sustained-release preparations and devices.

The active agent (e.g., a PPI modulator) may also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the active agent can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscan contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active agent (e.g., PPI modulator) which are adapted forthe extemporaneous preparation of sterile injectable or infusiblesolutions or dispersions, optionally encapsulated in liposomes. Theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. Optionally, the prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the PPImodulators above, as required, followed by filter sterilization. In thecase of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andthe freeze drying techniques, which yield a powder of the activeingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

For topical administration, active agents such as PPI modulators may beapplied in pure-form, i.e., when they are liquids. However, it willgenerally be desirable to administer them topically to the skin ascompositions, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

The PPI modulators can be applied topically to a subject's skin toreduce the size (and may include complete removal) of malignant orbenign growths. The PPI modulators can be applied directly to thegrowth. Preferably, the PPI is applied to the growth in a formulationsuch as an ointment, cream, lotion, solution, tincture, or the like.Drug delivery systems for delivery of pharmacological substances todermal lesions can also be used, such as that described in U.S. Pat. No.5,167,649 (Zook).

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcoholglycolblends, in which the active agent can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the active agent to the skin are disclosedin Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman (U.S.Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Accordingly, pharmaceutical compositions can comprise PPI modulator incombination with a pharmaceutically acceptable carrier. Pharmaceuticalcompositions adapted for oral, topical or parenteral administration,comprising an amount of an PPI modulator, constitute a preferredembodiment of the invention. The dose administered to a patient,particularly a human, in the context of the present invention should besufficient to achieve a therapeutic response in the patient over areasonable time frame, without lethal toxicity, and preferably causingno more than an acceptable level of side effects or morbidity. Oneskilled in the art will recognize that dosage will depend upon a varietyof factors including the condition (health) of the subject, the bodyweight of the subject, kind of concurrent treatment, if any, frequencyof treatment, therapeutic ratio, as well as the severity and stage ofthe pathological condition.

Depending upon the disorder or disease condition to be treated, asuitable dose(s) may be that amount that will reduce proliferation orgrowth of the target cell(s), or induce cell death. In the context ofcancer, a suitable dose(s) is that which will result in a concentrationof the active agent (e.g., one or more PPI modulators) in cancer tissue,such as a malignant tumor, which is known to achieve the desiredresponse. The preferred dosage is the amount which results in maximuminhibition of cancer cell growth, without unmanageable side effects.Administration active agents, such as PPI modulators, can be continuousor at distinct intervals, as can be determined by a person of ordinaryskill in the art.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsof the invention can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds of the invention based on the weight of the total compositionincluding carrier or diluents. Illustratively, dosage levels of theadministered active ingredients can be: intravenous, 0.01 to about 20mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 toabout 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 toabout 200 mg/kg, and preferably about 1 to 100 mg/kg; intranasalinstillation, 0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20mg/kg of animal (body) weight.

Mammalian species which benefit from the disclosed methods include, butare not limited to, primates, such as apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, andferrets; domesticated farm animals such as cows, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales. Other species that may benefit from the disclosed methodsinclude fish, amphibians, avians, and reptiles. As used herein, theterms “patient”, “subject”, and “individual” are used interchangeablyand are intended to include such human and non-human species. Likewise,in vitro methods of the present invention can be carried out on cells ofsuch human and non-human species. In some embodiments, the cells areobtained from a subject. In other embodiments, the cells are cells of acancer cell line.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical or veterinary professions, as appropriate.

The terms “cancer” and “malignancy” are used herein interchangeably torefer to or describe the physiological condition in mammals that istypically characterized by unregulated cell growth. The cancer may bemulti-drug resistant (MDR) or drug-sensitive. Examples of cancer includebut are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include breastcancer, prostate cancer, colon cancer, squamous cell cancer, small-celllung cancer, non-small cell lung cancer, gastrointestinal cancer,pancreatic cancer, cervical cancer, ovarian cancer, peritoneal cancer,liver cancer, e.g., hepatic carcinoma, bladder cancer, colorectalcancer, endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; uterine cancer; cancer of the urinary system, as well as othercarcinomas and sarcomas. Examples of cancer types that may potentiallybe sampled and treated using the kits and methods of the invention arealso listed in Table 1.

TABLE 1 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultHairy Cell Leukemia Acute Lymphoblastic Leukemia, Head and Neck CancerChildhood Hepatocellular (Liver) Cancer, Adult Acute Myeloid Leukemia,Adult (Primary) Acute Myeloid Leukemia, Childhood Hepatocellular (Liver)Cancer, Childhood Adrenocortical Carcinoma (Primary) AdrenocorticalCarcinoma, Childhood Hodgkin's Lymphoma, Adult AIDS-Related CancersHodgkin's Lymphoma, Childhood AIDS-Related Lymphoma Hodgkin's LymphomaDuring Pregnancy Anal Cancer Hypopharyngeal Cancer Astrocytoma,Childhood Cerebellar Hypothalamic and Visual Pathway Glioma,Astrocytoma, Childhood Cerebral Childhood Basal Cell CarcinomaIntraocular Melanoma Bile Duct Cancer, Extrahepatic Islet Cell Carcinoma(Endocrine Pancreas) Bladder Cancer Kaposi's Sarcoma Bladder Cancer,Childhood Kidney (Renal Cell) Cancer Bone Cancer, Osteosarcoma/MalignantKidney Cancer, Childhood Fibrous Histiocytoma Laryngeal Cancer BrainStem Glioma, Childhood Laryngeal Cancer, Childhood Brain Tumor, AdultLeukemia, Acute Lymphoblastic, Adult Brain Tumor, Brain Stem Glioma,Leukemia, Acute Lymphoblastic, Childhood Childhood Leukemia, AcuteMyeloid, Adult Brain Tumor, Cerebellar Astrocytoma, Leukemia, AcuteMyeloid, Childhood Childhood Leukemia, Chronic Lymphocytic Brain Tumor,Cerebral Leukemia, Chronic Myelogenous Astrocytoma/Malignant Glioma,Leukemia, Hairy Cell Childhood Lip and Oral Cavity Cancer Brain Tumor,Ependymoma, Childhood Liver Cancer, Adult (Primary) Brain Tumor,Medulloblastoma, Liver Cancer, Childhood (Primary) Childhood LungCancer, Non-Small Cell Brain Tumor, Supratentorial Primitive LungCancer, Small Cell Neuroectodermal Tumors, Childhood Lymphoma,AIDS-Related Brain Tumor, Visual Pathway and Lymphoma, Burkitt'sHypothalamic Glioma, Childhood Lymphoma, Cutaneous T-Cell, see MycosisBrain Tumor, Childhood Fungoides and Sézary Syndrome Breast CancerLymphoma, Hodgkin's, Adult Breast Cancer, Childhood Lymphoma, Hodgkin's,Childhood Breast Cancer, Male Lymphoma, Hodgkin's During PregnancyBronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, Adult ChildhoodLymphoma, Non-Hodgkin's, Childhood Burkitt's Lymphoma Lymphoma,Non-Hodgkin's During Carcinoid Tumor, Childhood Pregnancy CarcinoidTumor, Gastrointestinal Lymphoma, Primary Central Nervous SystemCarcinoma of Unknown Primary Macroglobulinemia, Waldenström's CentralNervous System Lymphoma, Malignant Fibrous Histiocytoma of PrimaryBone/Osteosarcoma Cerebellar Astrocytoma, Childhood Medulloblastoma,Childhood Cerebral Astrocytoma/Malignant Melanoma Glioma, ChildhoodMelanoma, Intraocular (Eye) Cervical Cancer Merkel Cell CarcinomaChildhood Cancers Mesothelioma, Adult Malignant Chronic LymphocyticLeukemia Mesothelioma, Childhood Chronic Myelogenous Leukemia MetastaticSquamous Neck Cancer with Chronic Myeloproliferative Disorders OccultPrimary Colon Cancer Multiple Endocrine Neoplasia Syndrome, ColorectalCancer, Childhood Childhood Cutaneous T-Cell Lymphoma, see MultipleMyeloma/Plasma Cell Neoplasm Mycosis Fungoides and Sézary MycosisFungoides Syndrome Myelodysplastic Syndromes Endometrial CancerMyelodysplastic/Myeloproliferative Diseases Ependymoma, ChildhoodMyelogenous Leukemia, Chronic Esophageal Cancer Myeloid Leukemia, AdultAcute Esophageal Cancer, Childhood Myeloid Leukemia, Childhood AcuteEwing's Family of Tumors Myeloma, Multiple Extracranial Germ Cell Tumor,Myeloproliferative Disorders, Chronic Childhood Nasal Cavity andParanasal Sinus Cancer Extragonadal Germ Cell Tumor NasopharyngealCancer Extrahepatic Bile Duct Cancer Nasopharyngeal Cancer, ChildhoodEye Cancer, Intraocular Melanoma Neuroblastoma Eye Cancer,Retinoblastoma Non-Hodgkin's Lymphoma, Adult Gallbladder CancerNon-Hodgkin's Lymphoma, Childhood Gastric (Stomach) Cancer Non-Hodgkin'sLymphoma During Pregnancy Gastric (Stomach) Cancer, Childhood Non-SmallCell Lung Cancer Gastrointestinal Carcinoid Tumor Oral Cancer, ChildhoodGerm Cell Tumor, Extracranial, Oral Cavity Cancer, Lip and ChildhoodOropharyngeal Cancer Germ Cell Tumor, ExtragonadalOsteosarcoma/Malignant Fibrous Germ Cell Tumor, Ovarian Histiocytoma ofBone Gestational Trophoblastic Tumor Ovarian Cancer, Childhood Glioma,Adult Ovarian Epithelial Cancer Glioma, Childhood Brain Stem OvarianGerm Cell Tumor Glioma, Childhood Cerebral Ovarian Low MalignantPotential Tumor Astrocytoma Pancreatic Cancer Glioma, Childhood VisualPathway and Pancreatic Cancer, Childhood Hypothalamic Pancreatic Cancer,Islet Cell Skin Cancer (Melanoma) Paranasal Sinus and Nasal CavityCancer Skin Carcinoma, Merkel Cell Parathyroid Cancer Small Cell LungCancer Penile Cancer Small Intestine Cancer Pheochromocytoma Soft TissueSarcoma, Adult Pineoblastoma and Supratentorial Primitive Soft TissueSarcoma, Childhood Neuroectodermal Tumors, Childhood Squamous CellCarcinoma, see Skin Pituitary Tumor Cancer (non-Melanoma) Plasma CellNeoplasm/Multiple Myeloma Squamous Neck Cancer with OccultPleuropulmonary Blastoma Primary, Metastatic Pregnancy and Breast CancerStomach (Gastric) Cancer Pregnancy and Hodgkin's Lymphoma Stomach(Gastric) Cancer, Childhood Pregnancy and Non-Hodgkin's LymphomaSupratentorial Primitive Primary Central Nervous System LymphomaNeuroectodermal Tumors, Childhood Prostate Cancer T-Cell Lymphoma,Cutaneous, see Rectal Cancer Mycosis Fungoides and Sézary Renal Cell(Kidney) Cancer Syndrome Renal Cell (Kidney) Cancer, ChildhoodTesticular Cancer Renal Pelvis and Ureter, Transitional Cell Thymoma,Childhood Cancer Thymoma and Thymic Carcinoma Retinoblastoma ThyroidCancer Rhabdomyosarcoma, Childhood Thyroid Cancer, Childhood SalivaryGland Cancer Transitional Cell Cancer of the Renal Salivary GlandCancer, Childhood Pelvis and Ureter Sarcoma, Ewing's Family of TumorsTrophoblastic Tumor, Gestational Sarcoma, Kaposi's Unknown Primary Site,Carcinoma of, Sarcoma, Soft Tissue, Adult Adult Sarcoma, Soft Tissue,Childhood Unknown Primary Site, Cancer of, Sarcoma, Uterine ChildhoodSezary Syndrome Unusual Cancers of Childhood Skin Cancer (non-Melanoma)Ureter and Renal Pelvis, Transitional Skin Cancer, Childhood Cell CancerUrethral Cancer Uterine Cancer, Endometrial Uterine Sarcoma VaginalCancer Visual Pathway and Hypothalamic Glioma, Childhood Vulvar CancerWaldenström's Macroglobulinemia Wilms' Tumor

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor or non-solid tumor. The solid tumormass, if present, may be a primary tumor mass. A primary tumor massrefers to a growth of cancer cells in a tissue resulting from thetransformation of a normal cell of that tissue. In most cases, theprimary tumor mass is identified by the presence of a cyst, which can befound through visual or palpation methods, or by irregularity in shape,texture or weight of the tissue. However, some primary tumors are notpalpable and can be detected only through medical imaging techniquessuch as X-rays (e.g., mammography) or magnetic resonance imaging (MRI),or by needle aspirations. The use of these latter techniques is morecommon in early detection. Molecular and phenotypic analysis of cancercells within a tissue can usually be used to confirm if the cancer isendogenous to the tissue or if the lesion is due to metastasis fromanother site. The treatment methods of the invention can be utilized forearly, middle, or late stage disease, and acute or chronic disease.

According to methods of the subject invention, a PPI modulator can beadministered to a patient by itself, or co-administered with one or moreother agents such as another PPI modulator, or a different agent oragents. Co-administration can be carried out simultaneously (in the sameor separate formulations) or consecutively. Furthermore, according tothe method of the subject invention, PPI modulators can be administeredto a patient as adjuvant therapy. For example, PPI modulators can beadministered to a patient in conjunction with chemotherapy, radiationtherapy, surgery, or a combination of two or more of the foregoing.

Thus, the PPI modulators, whether administered separately, or as apharmaceutical composition, can include various other components asadditives. Examples of acceptable components or adjuncts which can beemployed in relevant circumstances include antioxidants, free radicalscavenging agents, peptides, growth factors, antibiotics, bacteriostaticagents, immunosuppressives, anticoagulants, buffering agents,anti-inflammatory agents, anti-angiogenics, anti-pyretics, time-releasebinders, anesthetics, steroids, and corticosteroids. Such components canprovide additional therapeutic benefit, act to affect the therapeuticaction of the compounds of the invention, or act towards preventing anypotential side effects which may be posed as a result of administrationof the compounds. The PPI modulators can be conjugated to a therapeuticagent, as well.

Additional agents that can be co-administered to target cells in vitroor in vivo, such as in a patient, in the same or as a separateformulation, include those that modify a given biological response, suchas immunomodulators. For example, proteins such as tumor necrosis factor(TNF), interferon (such as alpha-interferon and beta-interferon), nervegrowth factor (NGF), platelet derived growth factor (PDGF), and tissueplasminogen activator can be administered. Biological responsemodifiers, such as lymphokines, interleukins (such as interleukin-1(IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), or other growth factors can be administered.In one embodiment, the methods and compositions of the inventionincorporate one or more agents selected from the group consisting ofanti-cancer agents, cytotoxic agents, chemotherapeutic agents,anti-signaling agents, and anti-angiogenic agents.

In some embodiments of the methods of the invention, at least oneadditional anti-cancer agent (e.g., a chemotherapeutic agent) isadministered with the PPI modulator. In some embodiments, theanti-cancer agent is selected from among suberoylanilide hydroxamic acid(SAHA) or other histone deacetylase inhibitor, arsenic trioxide,doxorubicin or other anthracycline DNA intercalating agent, andetoposide or other topoisomerase II inhibitor.

Within certain aspects of the present invention, one or more PPImodulators as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or more PPImodulators in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. A PPI modulator may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration. For certain topical applications,formulation as a cream or lotion, using well known components, ispreferred.

Various techniques may be utilized to facilitate delivery of the PPImodulators to the target cells in vitro (including ex vivo) and in vivo(Cellular Drug Delivery: Principles and Practice, edited by Lu, D. R.and Oie, S., Human Press, Totowa, N.J., 2004). Optionally, it may bedesirable to facilitate delivery of the PPI modulators through the outercell membrane. Various carrier molecules may be coupled to the PPImodulators to assist penetration through biological membranes. Forexample, small regions (e.g., 9-16 amino acids) of proteins calledprotein transduction domains (PTDs) cell penetrating peptides (CPP)possess the ability to traverse biological membranes through proteintransduction (Barnett, E. M. et al., Invest. Opthalmol. Vis. Sci., 2006,47:2589-2595; Schwarze S. R. et al., Science, 1999, 285(5433):1569-1572;Wadia, J. S, and Dowdy, S. F., Advanced Drug Delivery Reviews, 2005,57(4): 579-596; Wadia, J. S. and Dowdy, S. F., Curr. Opin. Biotechnol.,2002, 13(1)52-56; Ho A. et al., Cancer Research, 2001, 61:474-477;Futaki et al., J. Biol. Chem., 2001, February, 276(8):5836-5840; Cao G.et al., J. Neurosci., 2002, 22(13):5423-5431; Becker-Hapk, M. et al.,Methods, 2001, 24:247-256; Snyder, E. L. and Dowdy, S. F., Curr. Opin.Mol. Ther., 2001, 3:147-152; Lewin, M. et al., Nat. Biotechnol., 2000,18:410-414; Tung, C. H. et al., Bioorg. Med. Chem., 2002, 10:3609-3614;Richard, J. P., et al., J. Biol. Chem., Oct. 30, 2002, epub ahead ofprint). Transduction can occur in a receptor- andtransporter-independent fashion that appears to target the lipid bilayerdirectly. Proteins (peptides) and compounds that are linked to PTDs(e.g., covalently) have the capability to traverse outer cell membranes.Preferably, the delivery peptide is a trans-activating transcriptionalactivator (TAT) peptide or an Antennapedia (ANT) peptide, or aderivative of either. PTDs can be linked to the active agents fortransport across the cell membrane. One well characterized PTD is thehuman immunodeficient virus (HIV)-1 Tat peptide (see, for example, U.S.Pat. Nos. 5,804,604; 5,747,641; 5,674,980; 5,670,617; and 5,652,122).Peptides such as the homeodomain of Drosophila antennapedia (ANTP) andarginine-rich peptides display similar properties can be employed. VP22,a tegument protein from Herpes simplex virus type 1 (HSV-1), also hasthe ability to transport proteins across a cell membrane, and may becoupled to some PPIs.

DEFINITIONS

The terms “proximity probe” and “PLA probe” are used interchangeablyherein to refer to a moiety that binds to a target molecule, such as aprotein, in a sample and is detectable using oligonucleotideamplification methods. In some embodiments, a proximity probe comprisesa target molecule recognition moiety and an oligonucleotide probe. Insome embodiments, a target molecule specific amplification product canbe formed when at least two proximity probes specific for the targetmolecule are bound to the target molecule and the oligonucleotide probeof each proximity probe is ligated to one another to form a ligatedprobe that is amplified.

Polymerase chain reaction (PCR) is a process for amplifying one or moretarget nucleic acid sequences present in a nucleic acid sample usingprimers and agents for polymerization and then detecting the amplifiedsequence. The extension product of one primer when hybridized to theother becomes a template for the production of the desired specificnucleic acid sequence, and vice versa, and the process is repeated asoften as is necessary to produce the desired amount of the sequence. Theskilled artisan to detect the presence of desired sequence (U.S. Pat.No. 4,683,195) routinely uses polymerase chain reaction.

A specific example of PCR that is routinely performed by the skilledartisan to detect desired sequences is reverse transcript PCR (RT-PCR;Saiki et al., Science, 1985, 230:1350; Scharf et al., Science, 1986,233:1076). RT-PCR involves isolating total RNA from biological fluid,denaturing the RNA in the presence of primers that recognize the desirednucleic acid sequence, using the primers to generate a cDNA copy of theRNA by reverse transcription, amplifying the cDNA by PCR using specificprimers, and detecting the amplified cDNA by electrophoresis or othermethods known to the skilled artisan.

As used herein, the terms “label” and “tag” refer to substances that mayconfer a detectable signal (e.g., a signal representing theamplification product of a proximity ligation assay). A number oftechniques for visualizing or detecting labeled nucleic acids arereadily available. Such techniques include, microscopy, arrays,Fluorometry, Light cyclers or other real time PCR machines, FACSanalysis, scintillation counters, Phosphoimagers, Geiger counters, MRI,CAT, antibody-based detection methods (Westerns, immunofluorescence,immunohistochemistry), histochemical techniques, HPLC, spectroscopy,capillary gel electrophoresis, spectroscopy; mass spectroscopy;radiological techniques; and mass balance techniques.

As used herein, the term “ligand” refers to a molecule that contains astructural portion that is bound by specific interaction with aparticular receptor protein.

As used herein, the term “PPI” or “protein-protein interaction” refersto refers to the binding of two or more proteins together. PPIs may bebinary (two protein binding partners; a dimer) or tertiary (three ormore protein binding partners, e.g., a trimer). Proteins within a PPI(i.e., binding partners) may be the same protein (such as a homodimer orhomotrimer) or different proteins (such as a heterodimer orheterotrimer). Proteins within a tertiary interaction may be bound toone or more proteins within the PPI. In some embodiments, the PPIcomprises a tyrosine kinase, such as the human tyrosine kinases listedin Tables 3 and 4. The PPI may comprise an interaction between thebinding partners listed in Table 5 (human tyrosine kinase interactions).

In some embodiments of the methods and kits of the invention, at leastone of the target binding partners is of an RTK class selected fromamong RTK class I (EGF receptor family; ErbB family), RTK class II(insulin receptor family), RTK class III (PDGF receptor family), RTKclass IV (FGF receptor family) RTK class V (VEGF receptors family), RTKclass VI (HGF receptor family), RTK class VII (Trk receptor family), RTKclass VIII (Eph receptor family), RTK class IX (AXL receptor family),RTK class X (LTK receptor family), RTK class XI (TIE receptor family),RTK class XII (ROR receptor family), XIII (DDR receptor family), RTKclass XIV (RET receptor family), RTK class XV (KLG receptor family), RTKclass XVI (RYK receptor family), and RTK class XVII (MuSK receptorfamily).

As used herein, the term “PPI profile” or “protein-protein interactionprofile” refers to the result or output of an assay that measures therelative abundance of a protein-protein interaction. PPI profiles (e.g.,sample PPI profile, and reference PPI profile) may each be expressed asa value representative of the abundance of target binding partners inproximity to each other within the sample. The sample PPI profile andreference PPI profile may be expressed by any method useful forcomparison purposes, such as a numeric value, score, cutoff (threshold),or other expression. For example, a negative result in which a PPIprofile in a sample does not reach the cutoff would be useful clinicallyto avoid giving patients unnecessary PPI modulator therapy. A positiveresult in which a PPI profile in a sample is at or above the cutoffcould indicate potential sensitivity and allow the clinician to give thePPI modulator therapy to those patients who would be most likely tobenefit. Typically, a reference PPI profile is the PPI profile of aknown cancer cell (e.g., cancer cell of a known type or subtype) or of anormal cell (non-cancerous cell) useful for comparison purposes. Thereference PPI profile may also be a sample PPI profile obtained at adifferent time point than the sample PPI profile in question (before orafter, to observe changes to the PPI profile of a cancer over time).

As used herein, the term “PPI modulator” refers to an agent (e.g., smallmolecule, protein, nucleic acid) that directly or indirectly promotes,inhibits, or otherwise alters interaction between two or more proteins.The PPI modulator may be an inducer of the PPI (directly or indirectlypromoting interaction), or an inhibitor of the PPI (directly orindirectly inhibiting interaction). For example, a PPI inhibitor may bea kinase inhibitor, which indirectly acts to inhibit PPI.

As used herein, the term “bind” refers to any physical attachment orclose association, which may be permanent or temporary. The binding canresult from hydrogen bonding, hydrophobic forces, van der Waals forces,covalent, or ionic bonding, for example.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions (fragments) of immunoglobulinmolecules, i.e., molecules that contain an antibody combining site orparatope. The term is inclusive of monoclonal antibodies and polyclonalantibodies.

As used herein, the terms “administering” or “administer” are usedherein to refer the introduction of a substance into cells in vitro orinto the body of an individual in vivo by any route (for example, oral,nasal, ocular, rectal, vaginal and parenteral routes). Active agentssuch as PPI modulators may be administered individually or incombination with other agents via any route of administration, includingbut not limited to subcutaneous (SQ), intramuscular (IM), intravenous(IV), intraperitoneal (IP), intradennal (ID), via the nasal, ocular ororal mucosa (IN), or orally. For example, active agents such as PPImodulators can be administered by direct injection into or on a tumor,or systemically (e.g., into the circulatory system), to kill circulatingtumor cells (CTC).

As used herein, the term “polypeptide” refers to a sequence of two ormore amino acids, and is used interchangeably herein with the terms“protein”, “gene product”, “oligopeptide”, and “peptide”.

In the context of the instant invention, the terms “oligopeptide”,“polypeptide”, “peptide” and “protein” can be used interchangeably torefer to amino acid sequences of any length; however, it should beunderstood that the invention does not relate to the peptides in naturalform, that is to say that they are not in their natural environment butthat the peptide may have been isolated or obtained by purification fromnatural sources or obtained from host cells prepared by geneticmanipulation (e.g., the peptides, or fragments thereof, arerecombinantly produced by host cells, or by chemical synthesis). PeptidePPI modulators may also contain non-natural amino acids, as will bedescribed below. The terms “oligopeptide”, “polypeptide”, “peptide” and“protein” are also used, in the instant specification, to designate aseries of residues of any length, typically L-amino acids, connected oneto the other, typically by peptide bonds between the α-amino andcarboxyl groups of adjacent amino acids. Linker elements can be joinedto the peptides, for example, through peptide bonds or via chemicalbonds (e.g., heterobifunctional chemical linker elements) as set forthbelow. Additionally, the terms “amino acid(s)” and “residue(s)” can beused interchangeably.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer or other proliferation disorder. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. For example, treatment with active agent such as a PPImodulator may include reduction of undesirable cell proliferation,and/or induction of apoptosis and cytotoxicity. “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented or onset delayed. Optionally, the patient may be identified(e.g., diagnosed) as one suffering from the disease or condition (e.g.,proliferation disorder) prior to administration of the PPI modulator.

As used herein, the term “(therapeutically) effective amount” refers toan amount of the PPI modulator or other active agent (e.g., a drug)effective to treat a disease or disorder in a mammal. In the case ofcancer or other proliferation disorder, the therapeutically effectiveamount of the agent may reduce (i.e., slow to some extent and preferablystop) unwanted cellular proliferation; reduce the number of cancercells; reduce the tumor size; inhibit (i.e., slow to some extent andpreferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; reduce signaling inthe target cells, and/or relieve, to some extent, one or more of thesymptoms associated with the cancer. To the extent the administered PPImodulator prevents growth of and/or kills existing cancer cells, it maybe cytostatic and/or cytotoxic. For cancer therapy, efficacy can, forexample, be measured by assessing the time to disease progression (TTP)and/or determining the response rate (RR).

Treatments can be given in a growth inhibitory amount. As used herein,the term “growth inhibitory amount” of the active agent (e.g., PPImodulator) refers to an amount which inhibits growth or proliferation ofa target cell, such as a tumor cell, either in vitro or in vivo,irrespective of the mechanism by which cell growth is inhibited (e.g.,by cytostatic properties, cytotoxic properties, etc.). In a preferredembodiment, the growth inhibitory amount inhibits (i.e., slows to someextent and preferably stops) proliferation or growth of the target cellin vivo or in cell culture by greater than about 20%, preferably greaterthan about 50%, most preferably greater than about 75% (e.g., from about75% to about 100%).

The terms “cell” and “cells” are used interchangeably herein and areintended to include either a single cell or a plurality of cells, invitro or in vivo, unless otherwise indicated.

As used herein, the term “sample” refers a biological composition thatpotentially contains target molecules (e.g., one or more protein bindingpartners in a protein-protein interaction of interest). Preferably, thesample is a cellular sample (samples of intact cells, e.g., a cytologysample). One or more samples of a malignancy may be obtained from asubject by techniques known in the art, such as biopsy. The type ofbiopsy utilized is dependent upon the anatomical location from which thesample is to be obtained. Examples include fine needle aspiration (FSA),excisional biopsy, incisional biopsy, colonoscopic biopsy, punch biopsy,and bone marrow biopsy. Depending on the PPI of interest, the sample maybe obtained from a pleural effusion or the bloodstream, for example. Itshould be understood that the methods of the invention may include astep in which a sample is obtained directly from a subject;alternatively, a sample may be obtained or otherwise provided, e.g., bya third party.

A sample may be taken from a subject having or suspected of havingcancer. A sample may also comprise proteins isolated from a tissue orcell sample from a subject. In certain aspects, the sample can be, butis not limited to tissue (e.g., biopsy, particularly fine needle biopsy,excision, or punch biopsy), blood, serum, plasma. The sample can befresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffinembedded) tissues or cells (e.g., FFPE tissue). In a particular aspect,the sample is a sample of lung cancer cells, colon cancer cells, breastcancer cells, ovarian cancer cells, renal cancer cells, melanoma cells,prostate cancer cells, CNS cancer cells, or leukemia cells, esophagealcancer cells, stomach cancer cells, bile duct cancer cells, liver cancercells, cancer cells of the rectum or anus, lymphoma, leukemia, cervicalcancer cells, bladder cancer cells, or protein from any of theaforementioned cancers. Depending upon the cancer type, the cancer cellsmay be circulating tumor cells (CTCs).

As used herein, the term “anti-cancer agent” refers to a substance ortreatment (e.g., agent or radiation therapy) that inhibits the functionof cancer cells, inhibits their growth, formation, and/or causes theirdestruction in vitro or in vivo. Examples include, but are not limitedto, cytotoxic agents (e.g., 5-fluorouracil, TAXOL), chemotherapeuticagents, and anti-signaling agents (e.g., the PI3K inhibitor LY).Anti-cancer agents include but are not limited to the chemotherapeuticagents listed in Table 2, and other agents disclosed herein, such aserlotinib or other small molecule epidermal growth factor tyrosinekinase inhibitors (EGFR TKI). In the kits and methods of the invention,the treatment or potential treatment may be an anti-cancer agent (forexample, a PPI modulator such as a kinase inhibitor or other PPIinhibitor).

As used herein, the term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells in vitro and/or in vivo. The term is intended to includeradioactive isotopes (e.g., At²¹¹, ¹³¹I, ¹²⁵I, ⁹⁰Y, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents,toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, and antibodies, includingfragments and/or variants thereof.

As used herein, the term “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer, such as, for example, taxanes, e.g.,paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,vincristine, vinblastine, anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc.Examples of chemotherapeutic agents that may be used in conjunction withPPI modulators are listed in Table 2. In a preferred embodiment, thechemotherapeutic agent is one or more anthracyclines. Anthracyclines area family of chemotherapy drugs that are also antibiotics. Theanthracyclines act to prevent cell division by disrupting the structureof the DNA and terminate its function by: (1) intercalating into thebase pairs in the DNA minor grooves; and (2) causing free radical damageof the ribose in the DNA. The anthracyclines are frequently used inleukemia therapy. Examples of anthracyclines include daunorubicin(CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX), epirubicin (ELLENCE,PHARMORUBICIN), and idarubicin (IDAMYCIN).

TABLE 2 Examples of Chemotherapeutic Agents 13-cis-Retinoic Acid Mylocel2-Amino-6- Letrozole Mercaptopurine Neosar 2-CdA Neulasta2-Chlorodeoxyadenosine Neumega 5-fluorouracil Neupogen 5-FU Nilandron6-TG Nilutamide 6-Thioguanine Nitrogen Mustard 6-Mercaptopurine Novaldex6-MP Novantrone Accutane Octreotide Actinomycin-D Octreotide acetateAdriamycin Oncospar Adrucil Oncovin Agrylin Ontak Ala-Cort OnxalAldesleukin Oprevelkin Alemtuzumab Orapred Alitretinoin OrasoneAlkaban-AQ Oxaliplatin Alkeran Paclitaxel All-transretinoic acidPamidronate Alpha interferon Panretin Altretamine ParaplatinAmethopterin Pediapred Amifostine PEG Interferon AminoglutethimidePegaspargase Anagrelide Pegfilgrastim Anandron PEG-INTRON AnastrozolePEG-L-asparaginase Arabinosylcytosine Phenylalanine Mustard Ara-CPlatinol Aranesp Platinol-AQ Aredia Prednisolone Arimidex PrednisoneAromasin Prelone Arsenic trioxide Procarbazine Asparaginase PROCRIT ATRAProleukin Avastin Prolifeprospan 20 with Carmustine implant BCGPurinethol BCNU Raloxifene Bevacizumab Rheumatrex Bexarotene RituxanBicalutamide Rituximab BiCNU Roveron-A (interferon alfa-2a) BlenoxaneRubex Bleomycin Rubidomycin hydrochloride Bortezomib SandostatinBusulfan Sandostatin LAR Busulfex Sargramostim C225 Solu-Cortef CalciumLeucovorin Solu-Medrol Campath STI-571 Camptosar StreptozocinCamptothecin-11 Tamoxifen Capecitabine Targretin Carac Taxol CarboplatinTaxotere Carmustine Temodar Carmustine wafer Temozolomide CasodexTeniposide CCNU TESPA CDDP Thalidomide CeeNU Thalomid CerubidineTheraCys cetuximab Thioguanine Chlorambucil Thioguanine TabloidCisplatin Thiophosphoamide Citrovorum Factor Thioplex CladribineThiotepa Cortisone TICE Cosmegen Toposar CPT-11 TopotecanCyclophosphamide Toremifene Cytadren Trastuzumab Cytarabine TretinoinCytarabine liposomal Trexall Cytosar-U Trisenox Cytoxan TSPA DacarbazineVCR Dactinomycin Velban Darbepoetin alfa Velcade Daunomycin VePesidDaunorubicin Vesanoid Daunorubicin Viadur hydrochloride VinblastineDaunorubicin liposomal Vinblastine Sulfate DaunoXome Vincasar PfsDecadron Vincristine Delta-Cortef Vinorelbine Deltasone Vinorelbinetartrate Denileukin diftitox VLB DepoCyt VP-16 Dexamethasone VumonDexamethasone acetate Xeloda dexamethasone sodium Zanosar phosphateZevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronic acid DICZometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSF DoxorubicinGoserelin Doxorubicin liposomal granulocyte - colony stimulating factorDroxia Granulocyte macrophage colony stimulating DTIC factor DTIC-DomeHalotestin Duralone Herceptin Efudex Hexadrol Eligard Hexalen EllenceHexamethylmelamine Eloxatin HMM Elspar Hycamtin Emcyt Hydrea EpirubicinHydrocort Acetate Epoctin alfa Hydrocortisone Erbitux Hydrocortisonesodium phosphate Erwinia L-asparaginase Hydrocortisone sodium succinateEstramustine Hydrocortone phosphate Ethyol Hydroxyurea EtopophosIbritumomab Etoposide Ibritumomab Tiuxetan Etoposide phosphate IdamycinEulexin Idarubicin Evista Ifex Exemestane IFN-alpha Fareston IfosfamideFaslodex IL-2 Femara IL-11 Filgrastim Imatinib mesylate FloxuridineImidazole Carboxamide Fludara Interferon alfa Fludarabine InterferonAlfa-2b (PEG conjugate) Fluoroplex Interleukin-2 FluorouracilInterleukin-11 Fluorouracil (cream) Intron A (interferon alfa-2b)Fluoxymesterone Leucovorin Flutamide Leukeran Folinic Acid Leukine FUDRLeuprolide Fulvestrant Leurocristine G-CSF Leustatin Gefitinib LiposomalAra-C Gemcitabine Liquid Pred Gemtuzumab ozogamicin Lomustine GemzarL-PAM Gleevec L-Sarcolysin Lupron Meticorten Lupron Depot MitomycinMatulane Mitomycin-C Maxidex Mitoxantrone Mechlorethamine M-PrednisolMechlorethamine MTC Hydrochlorine MTX Medralone Mustargen Medrol MustineMegace Mutamycin Megestrol Myleran Megestrol Acetate Iressa MelphalanIrinotecan Mercaptopurine Isotretinoin Mesna Kidrolase Mesnex LanacortMethotrexate L-asparaginase Methotrexate Sodium LCR Methylprednisolone

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid tumor mass. A primary tumor mass refers to agrowth of cancer cells in a tissue resulting from the transformation ofa normal cell of that tissue. In most cases, the primary tumor mass isidentified by the presence of a cyst, which can be found through visualor palpation methods, or by irregularity in shape, texture, or weight ofthe tissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site. The peptide PPI modulators may be capable of inducingapoptosis in tumor cells, reducing tumor size, and/or inhibiting tumorcell growth. The peptides of the invention (or nucleic acids encodingthem) can be administered locally at the site of a tumor (e.g., bydirect injection) or remotely. The peptide PPI modulators may inducecell death in circulating tumor cells (CTC) in a subject, e.g., byadministering the peptides or encoding nucleic acids intravenously.Furthermore, the peptide PPI modulators may prevent or reduce onset ofmetastasis to other tissues, e.g., to the bone.

As used herein, the term “signaling” and “signaling transduction”represents the biochemical process involving transmission ofextracellular stimuli, via cell surface receptors through a specific andsequential series of molecules, to genes in the nucleus resulting inspecific cellular responses to the stimuli.

As used herein, the term “pharmaceutically acceptable salt or prodrug”is intended to describe any pharmaceutically acceptable form (such as anester, phosphate ester, salt of an ester or a related group) of a PPImodulator or other active agent, which, upon administration to asubject, provides the mature or base compound. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. Pharmaceutically acceptable prodrugsrefer to a compound that is metabolized, for example hydrolyzed oroxidized, in the host to form the compound of the present invention.Typical examples of prodrugs include compounds that have biologicallylabile protecting groups on a functional moiety of the active compound.Prodrugs include compounds that can be oxidized, reduced, aminated,deaminated, hydroxyl ated, dehydroxylated, hydrolyzed, dehydrolyzed,alkylated, dealkylated, acylated, deacylated, phosphorylated,dephosphorylated to produce the active compound.

The terms “link” or “join” refers to any method known in the art forfunctionally connecting peptides, including, without limitation,recombinant fusion, covalent bonding, disulfide bonding, ionic bonding,hydrogen bonding, and electrostatic bonding.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state.

As used in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a compound” includes more than onesuch compound. Reference to “PPI modulator” includes more than one suchPPI modulator. A reference to “an antibody” includes more than one suchantibody, and so forth.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification Principles and Practice (2nded., Springer-Verlag); and PCR: A Practical Approach (McPherson et al.Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

Experimental controls are considered fundamental in experiments designedin accordance with the scientific method. It is routine in the art touse experimental controls in scientific experiments to prevent factorsother than those being studied from affecting the outcome.

Materials and Methods

Primary Xenograft Mouse Models.

4-6 weeks old female hairless mice Crl:SHO-Prkdc-SCID-Hr-hr mice will bepurchased from approved vendors and housed in the institutional animalfacilities according to protocols set out by the American Associationfor Accreditation of Laboratory Animal Care. The inventor has activeIACUC protocols for establishing tumor xenografts in mice and to performdrug treatment experiments. Mice will be anesthetized with isofluorane,a small incision and a small subcutaneous pocket will be made in eachside of the lower back, and a piece of tumor collected from NSCLCpatients will be deposited in each pocket. Animals will be observeduntil palpable tumors are present and subsequently treated vehicle (31).The mice will be monitored 2-3 times per week. If any mice demonstratediscomfort, i.e., lethargy, abnormal posture, failure to groom, ruffled,matted or soiled hair coat, rapid, shallow and/or labored breathing,have tumors greater than 1.0 cm in diameter, have tumors which interferewith posture, locomotion or feeding, or have tumors abscess through theskin, they will be euthanized using CO₂. Each group of mice will be putin the cleaned and dried chamber, and then the chamber will be slowlyfilled by compressed carbon dioxide gas. Mice will be quickly euthanizedby carbon dioxide inhalation. Mouse death will be confirmed byverification of cessation of cardiovascular and respiratorymovementactivity. This method is consistent with recommendations of thePanel on Euthanasia of the American Veterinary Medical Association. Logentries for complications of either tumor growth or tumor metastasiswill be maintained. For tumor harvesting, mice will be euthanized by CO₂inhalation and tumors will be excised and snap frozen in liquidnitrogen.

Cell Line Xenograft Models.

Tumor tissues from subcutaneous mouse models of lung cancer will beused. CD-1 nu/nu mice do not have a thymus and are thereforeimmunodeficient and unable to produce T-cells. Mice have been selectedfor these experiments since they are most suitable for xenograft andorthographic studies of lung cancer, are readily available and notendangered, and can be safely maintained in the laboratory animalvivarium. CD-1 nu/nu mice will be purchased from approved vendors andhoused in institutional animal facilities according to protocols set outby the American Association for Accreditation of Laboratory Animal Care.The inventor currently has active IACUC protocol for establishingsubcutaneous tumor xenografts in mice. Both male and female mice ofapproximately six weeks of age will be used. Mice will be anesthetizedin an induction chamber containing 3% isoflurane and 97% oxygen. Micewill be subsequently shaved in the flank area and injected with varioushuman lung cancer cell lines. Animals will be observed until palpabletumors are present and subsequently treated vehicle or compounds in 0.2ml vehicle containing 0.5% methylcellulose and 0.4% polysorbate 80(Tween 80) (59, 60). The mice will be monitored 2-3 times per week. Ifany mice demonstrate discomfort, i.e., lethargy, abnormal posture,failure to groom, ruffled, matted or soiled hair coat, rapid, shallowand/or labored breathing, have tumors greater than 1.0 cm in diameter,have tumors which interfere with posture, locomotion or feeding, or havetumors abscess through the skin, they will be euthanized using CO2. Eachgroup of mice will be put in the cleaned and dried chamber, and then thechamber will be slowly filled by compressed carbon dioxide gas. Micewill be quickly euthanized by carbon dioxide inhalation. Mouse deathwill be confirmed by verification of cessation of cardiovascular andrespiratory movement/activity. This method is consistent withrecommendations of the Panel on Euthanasia of the American VeterinaryMedical Association. Log entries for complications of either tumorgrowth or tumor metastasis will be maintained. For tumor harvesting,mice will be euthanized by CO2 inhalation and tumors will be excised andsnap frozen in liquid nitrogen.

-   -   Patient consenting process: The TCC protocol was IRB approved in        January 2006; since then, 33,000+ patients have prospectively        consented to have their tissue collected and agreed to lifetime        follow-up. Moffitt expects 100,000 consents within 4 years at 15        partner institutions.    -   Sample Collection To date, over 10,000 tumors have been        collected. This number will grow exponentially in coming years.        TCC collects tumor, blood and urine samples as well as clinical        data such as risk factors, therapies and outcomes. All tissues        are snap frozen within 15 minutes of resection and 2D bar code        labeled. Moffitt has robotic biobanking and every tissue is        macrodissected to ≧85% purity.    -   Pathologic Review of Frozen Tissue Specimens: Each frozen tissue        specimen is subjected to comprehensive histopathologic review by        Board-certified Pathologists. Each tumor sample is quantified        for proportions of the following tissue components: malignant        (viable tumor cellularity, tumor necrosis and stroma), normal,        abnormal (including inflammation, ulceration, atrophy,        hyperplasia etc) and benign neoplastic tissues. In order to        achieve the highest possible purity of viable malignant tumor        tissue for each specimen, every effort is made to exclude        (macrodissect) the latter three components from the tissue        processed for subsequent molecular analyses. In order to ensure        the highest quality of tissue specimens and to control for        pre-analytic variables, the tissue collection and review        processes are regulated by specific Standard Operating        Procedures (SOPs) and QA/QC protocols.    -   Gene Expression Profiling: 7,000 samples have been analyzed to        date. Identifying molecular signatures is a key component of        precision-based personalized medicine. To date, TCC has focused        on gene expression but other modalities, including quantitative        proteomics, are planned.    -   Biorepository: All specimens collected under the TCC protocol        are stored in a single location at Moffitt in a biobank capable        of storing 1,000,000 samples. Currently, tumor specimens are        collected from patients with colon, lung, prostate, brain,        breast, pancreatic, ovarian, bladder, and renal cancers. As the        resource develops, tumor specimens from all disease sites will        be acquired from consortium members.    -   Data Warehouse Storage is provided for patient records, medical        history, registry data, molecular profiles (e.g. gene        expression), and clinicopathologic data in compliance with HIPAA        requirements. Within 5 years, the warehouse is expected to hold        information on primary and metastatic biopsies for over 50,000        patients. Appropriate access is given to the patient, the        clinician and researcher to lead to the creation of        evidence-based guidelines. Separate web portals are being        developed to meet these distinct needs.

Translational research is incorporated along this continuum of care andfollow-up. TCC is more than just a registry, cohort study, or tissuebank; the system prospectively consents patients, collects clinical datathroughout a patient's lifetime, creates gene expression profiles of alltumors, and enables access to the data for the patient, clinician andresearcher. Its capability for in-depth analysis could lead to improvingthe standard of care addressing each patient's specific needs.

Subjects are being recruited to Total Cancer Care™ (TCC) sites in FL(including the Moffitt Cancer Center (MCC), primary sponsor of thestudy), NC, SC, CT, KY, LA, IN and NE according to the TCC protocol andconsent. The total recruitment expected across all sites is by 2012 is100,000 patients. Patients ages 18 and older years with newly diagnosed,recurrent or metastatic cancers (stages I-IV) are being recuited.Inclusion criteria are broad to include any willing patient that hasvisited one of the consortium sites and presented with a cancerdiagnosis. Exclusion criteria is any registered patient who is notwilling or able to sign the consent for data collection. The TCC studyprovides for the collection of patient biospecimens and data throughoutthe patients life in a large prospective study. Tumor samples collectedeither through resection or biopsy for research studies are comprised ofexcess tissue not necessary for pathological diagnosis or clinical careand are banked by MCC. No appreciable risks to subjects associated withtissue collection and analysis is anticipated; the patient's surgeonwill remove this tissue as part of his or her clinical care, and noadditional tissue will be removed for the TCC Study unless specified andconsent granted by the patient prior to the procedure. Additionalbiospecimens, including blood and urine, maybe collected as described inthe TCC protocol and consent documents, although these are not used forthe study purposes of the proposed research described in thisapplication. If there is a change in protocol procedures or desiredbiospecimen types an amendment to the protocol and consent are providedto the University of South Florida's Internal Review Board (IRB) forconsideration of the benefits to research and the impact on patientcare, well being, harm and discomfort. Changes to the TCC protocol atConsortium sites are presented to local IRBs for consideration prior toadditional biospecimen collections. Subjects may feel uncomfortableanswering personal questions on study questionnaires or knowing thattheir medical records are being reviewed. Patients will be informed thatthat they may skip any questions they prefer not to answer. Great carewill be taken to protect patient privacy throughout the study inaccordance with MCC policies.

The inventor has an active protocol that also allows for patient chartreview to gather information necessary for this project as well asaccess tumor tissue for molecular assays. This study was approved by theUniversity of South Florida IRB and allows access to PHI for researchpurposes described in this proposal. This allows for chart review by theinventor and his trained staff to identify clinical variables such asage, sex, histology of tumor, stage, response to therapy, and overallsurvival.

Some of the Figures herein have been labeled with one or more colors tofacilitate interpretation when the Figures are viewed in black andwhite. The lack of a color label on any particular region of a figureshould not necessarily be construed as an absence of color in thatregion.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Epidermal Growth Factor Receptor (EGFR) Protein-ProteinInteractions as Biomarkers

Cancer is recognized to be a result of changes in cellular genomesresulting in aberrant signaling proteins causing deregulated cellgrowth, survival, and metastasis. These changes rewire entire signaling‘circuits’ resulting in aberrant growth and metastasis. Critical toprotein function and signaling is the formation of signaling complexesand networks of signaling proteins that act in concert to produce aphysiological signal (1, 2). State of the art mass spectrometry is nowable to accurately map protein-protein interaction (PPI) complexes andnetworks (3, 4). Initially, this was performed in yeast with high impactpapers demonstrating the modular organization of PPI and insight intocellular signaling mechanisms (5, 6). However, improvements in massspectrometry coupled with improved biochemical purification strategiesare now enabling PPI mapping in mammalian cells, including cancer cells(7-9). This allows a better understanding of how cancer proteins drive asignaling network to transform cells. The application of network theoryto biology may enable a better understanding of cancer, improve abilityto classify tumors, and suggest therapeutic approaches against cancer‘hub’ proteins or suggest rational combination approaches (10-14). Inaddition, comprehensive databases (such as www.hprd.org) now listprotein interactions based on manually annotated reviews of theliterature. Using these databases, groups can construct theoretical PPIthat could be important in diseases such as cancer. One group used thesedatabases to demonstrate the importance of deregulated PPI in aggressivecancers (15). Importantly, this network view can produce biomarkerscategorized by protein-protein interactions that carry information toguide clinical decision making. Despite the explosion of PPI datasets,most are limited in pre-clinical space and interrogations of PPI inhuman cancer specimens is lacking. Thus, the present inventors haveendeavored to make ‘network medicine’ a reality (16).

The present inventors have initiated a system wide and global analysisof the PPI driven by hyperactivated epidermal growth factor receptor(EGFR) resulting from somatic mutation in non-small cell lung cancer(NSCLC) (17-19). The inventors' approach has been to combine (i)affinity purification mass spectrometry (AP-MS) and (ii) phosphotyrosineproteomics. The inventors use cell models that harbor mutant EGFRproteins and are highly sensitive to EGFR TKI. As the network of EGFR ishighly dependent on cell context, this approach offers the best chanceof recovering pertinent proteins and mechanisms, a potential flaw inusing cells other than lung cancer cells driven by mutant EGFR. Taggedversions of EGFR are initially used to identify interactions and secondround of AP-MS is performed to more fully construct the network (20,21). The inventors simultaneously performed tyrosinephosphoproteomics—tyrosine phosphorylated (pTyr) peptides are enrichedusing an anti-phosphotyrosine antibody and then identified andquantified on an Orbitrap mass spectrometer using published methods (9).Using protein-protein interaction databases, the inventors linked pTyrcontaining proteins to proteins identified in AP-MS experiments. Thisproduced the final map of nearly 300 proteins (shown in FIG. 1). Thisinteraction network allows functional interrogation of targets importantin driving mutant EGFR growth and survival signals. In addition, theinteractions themselves can be used as biomarkers in human cancerspecimens by identifying an EGFR activating state. Thus, the criticalquestion becomes, how can one ‘translate’ these preclinical massspectrometry data into human cancer samples?

Translation of these network approaches to tumor samples is hampered bya number of challenges. First, almost all studies have been carried outin engineered mammalian cells that express a tagged version of theprotein of interest; this limits the ability to use these modern AP-MSapproaches in samples from patients. Second, most samples from patientsare formalin fixed and paraffin embedded. This precludes the ability touse fresh frozen tissue for immunoprecipitation and western blotting(IP-WB) to identify protein complexes. IP-WB also requires a largeamount of starting material that can be difficult to obtain with needlebiopsies.

One solution to mapping networks identified using MS-based proteomics isproximity ligation assays (PLA) (22-27). Briefly, two proteins incomplex are each identified with primary antibodies specific for theprotein and linked to a conjugated oligonucleotide. A connectoroligonucleotide links both proximity probes allowing ligation andformation of a template for PCR amplification. The resulting rollingcircular amplification (RCA) serves as a target for hybridizedfluorescently labeled detection oligonucleotides allowing distinct andbright spots to be identified and quantified in a fluorescentmicroscope. See http:www.olink.webbhuset.com/movie.php for an animationdescribing this technology. This technology has been used to identify incells in situ Myc-Max interactions, tyrosine phosphorylation of receptortyrosine kinases, and interactions of proteins in human tissue sections.

As little has been done to establish biomarker systems to measureprotein-protein based biomarkers in cancer, it would be advantageous tohave an assay (e.g., a PLA) that can quantitatively measure definedprotein-protein interactions in cancer specimens (e.g., EGFRprotein-protein interactions in lung cancer specimens) and relateexpression of these interactions to clinical outcome variables. Lungcancer accounts for over 160,000 deaths per year in the U.S., more thanbreast, colon, prostate and pancreatic cancer combined (28). The overallfive-year survival rate for lung cancer is approximately 15%, and unlikeother solid tumors such as colon or breast cancer, little progress hasbeen made in improving survival. There is therefore an important unmetneed to better identify key drivers of lung cancer that can betherapeutically exploited. Coupled with this need is the ability toidentify patients' subsets driven by drug targets. EGFR is selected as atest case to develop these assays based on (i) its known role in lungcancer pathogenesis, (ii) use of EGFR TKI for treatment of lung cancer,(iii) experimentally derived protein-protein interaction data derived inthe inventor's laboratory using mass spectrometry and (iv) availabilityof antibody reagents (29). The assays are not meant to replace existingclinical assays that predict EGFR kinase inhibitor sensitivity (i.e.,EGFR mutation analysis) but are meant to allow a starting point todevelop PPI based biomarkers that could be applied to other cancerpathways or drugs. This could allow markers that identify patientsbenefiting from EGFR TKI whose tumors do not harbor EGFR mutations.Finally, success here can be translated to other signaling systems, suchas MET and IGFR, where interactions could be informative (for example,MET with Gab1 and IGFR with IRS proteins) and biomarkers predictingresponse are unclear (30).

The approach to develop biomarkers based on protein-protein interactionsgoes one step beyond measurement of protein expression as it determinesthe binding to two proteins together in tumor tissues. Two proteins maybe equally expressed, but because of other nuances in the cancer cell,do not form a signaling complex that drives a signaling cascade. Inanother tumor cell, these proteins form a complex and drive a signalingcascade. Gene-based approaches that measure mRNA expression orimmunohistochemistry approaches that measure protein expression would beunable to discriminate these two cases. One aspect of the invention is atranslatable assay system measuring protein-protein interactions drivenby hyperactivated forms of EGFR and this approach will be tested inpanels of cell lines, animal tumor models, and specimens from lungcancer patients. The inventor builds on the AP-MS mass spectrometry datato build protein-protein interaction networks in lung cancer cell linesdriven by activated EGFR and translate these findings into patient basedmaterials. Four aims are described herein.

Aim 1: Establish and Validate Proximity Ligation Assays that MeasureEGFR Protein-Protein Interactions

The invention provides assays measuring protein-protein interactionsbased on proximity ligation assay (PLA) technology. This technology iscapable of detecting single protein events such as protein interactions.The assay provides exact spatial information on the location of theevents and an objective means of quantifying the events. Experimentallyderived mass spectrometry data defining interacting proteins within theEGFR network will guide selection of protein complexes for assaydevelopment.

Aim 2: Characterize EGFR protein interactions in cell and tumor modelswith known EGFR mutation status. Here the hypothesis is that cells andtumors with activating EGFR mutations will demonstrate constitutivecomplexes with EGFR proteins. This will be tested in both cell lineswith known EGFR mutation status and tumor samples from patients withknown EGFR mutation status. The inventor will also test the hypothesisthat the degree of EGFR complexes is related to sensitivity to EGFRtyrosine kinase inhibitors (TKI).

Aim 3: Characterize changes in EGFR protein-protein interactions inresponse to EGFR tyrosine kinase inhibitors in primary lung cancerxenograft models and patient samples. EGFR inhibitors are predicted todisrupt signaling complexes on the EGFR protein. The inventor will useprimary lung cancer xenograft systems and samples from patients beforeand after EGFR inhibitor therapy to measure differences in EGFRprotein-protein interactions.

Aim 4: Determine if EGFR protein-protein interactions are associatedwith responses to EGFR tyrosine kinase inhibitors in human lung cancersamples. The inventor hypothesizes that quantitative measurements ofEGFR protein-protein interactions could be used as biomarkers to predictactivity of EGFR kinase inhibitors in patients with advanced lungcancer. The inventor has developed PLA assays for EGFR protein-proteininteractions in lung cancer samples and related these measurements toresponse to EGFR inhibitors in a group of patients with advanced lungcancer.

Aim 1: Establish Proximity Ligation Assays that Measure EGFRProtein-Protein Interactions.

The mass spectrometry data provides a set of proteins physicallyinteracting with one another that can be translated into assays in tumormaterial. The initial set of proteins consisting of EGFR, Src, ErbB3,Mig6, Grb2, Sts1, and p85 is based on the MS data and the literature.Aside from EGFR, ErbB3 and p85 were selected as they are importantcomponents of mutant EGFR signaling that lead to activation of Aktsignaling and lung cancer survival (31, 32). Grb2 was chosen based onits role in activating Ras signaling downstream of EGFR (33, 34). Mig6and Sts1 were selected based on their ability to negatively regulateEGFR signaling (35-38). Src is intimately involved in EGFR signaling andimportant in EGFR driven lung cancer (39-41). The inventor will measurethe following six interactions: EGFR:ErbB3, EGFR:Grb2, EGFR:Mig6,EGFR:Sts1, EGFR:Src and ErbB3:p85. Guidelines for antibody validationdescribed by David Rimm (42) and summarized below will be followed.Protein extracts from a panel of lung cancer cell lines with and withoutEGFR mutations will be used to test for antibody specificity usingwestern blots. A panel of cells is examined to avoid reachingconclusions of specificity based on a single cell line. Antibodies thatappear specific (dominant single band, few other bands outside MW rangeof protein) will be selected for further development.

Immunofluorescence (IF) conditions can be optimized for each antibody,including a series of antibody dilutions, different sets of temperaturesand incubation times, and different concentrations of secondary antibodyconjugated with fluorescence dye. Patterns of IF staining will beexamined using fluorescence and confocal microscopy using PC9 cellsgrown on a 8 or 16 well chamber slides. The antibodies that pass thetest in this run of validation have to reach three criteria at the sametime: 1) the location of cell compartment must be consistent with thosethat previous reported (i.e., membranecytoplasmnuclear); 2) antibodiesmust be sensitive enough to give enough signal intensity abovebackground; 3) Every pair of antibodies must pass the double stainingtest under the same conditions which is fundamental to the PLA assay.Two antibodies from different species work must well under identicalconditions with balanced intensities thus requiring us to screendifferent pairs of antibodies based on the information from commercialsources and the IF results described above.

Validated antibodies will be used for PLA. An initial experiment wasperformed (FIGS. 3A-F). PC9 lung cancer cells harboring an activatingEGFR mutation were evaluated for EGFR:Grb2 binding based on the MS data.Antibodies were selected and validated with both western blotting and IFas described above. PLA was then tested to measure these interactions inuntreated cells and in cells treated with EGFR TKI erlotinib. Additionalcontrols were added such as leaving out either primary antibody orleaving out secondary antibodies. Strong foci were identified indicativeof EGFR:Grb2 interactions that are significantly reduced by erlotiniband completely absent in the negative controls. To assess non-specificnoise background in the PLA assays, at least four negative controls willbe used in parallel: omit one of the two antibodies in each well, andomit one of two secondary antibodies conjugated with PLUS or MINUS DNAfragments in each well. A positive control is also used in eachindividual test. Results above used a Leica TCS SP5 AOBS laser scanningconfocal microscope. Analysis of labeled foci in the maximum projectionimages was performed using Image Pro Plus version 6.2 (MediaCybernetics, Inc., Silver Springs, Md.). Regions of interest werederived manually and by image analysis using a click and growmethodology to define cell borders and segment individual cells.Histogram threshholding for the biomarker targeted channels were used toidentify the number of foci and determine the mean intensity of the focifor each individual cell. Foci are defined as any Alexa 555 labeledobject within the cell that has an area of at least 5 pixels overthreshold. Other approaches will be considered. PLA assay creates aperfect fluorescence signal allowing conventional IF to be adapted tothe mature AQUA system. The data collection with AQUA includes scanningfluorescence signal in the tissue section with AQUAsition, andtransferring image signals into digital numbers with AQUAanlysissoftware, which is the newest version package, AQUA 2.2. Definiens®Developer v1.2 (Definiens, Munich, Germany) software suite can be usedto automatically find individual cells and analyze them. A plasmamembrane or whole cytoplasm stain may need to be added to accomplishthis task. This will allow for batch processing and increase imageanalysis throughput significantly, while retaining quality controlstandards necessary for this project.

Aim 2: Characterize EGFR Protein Interactions in Cell and Tumor Modelswith Known EGFR Mutation Status.

The inventor proposes that cells and tumors with activating EGFRmutations will demonstrate constitutive complexes with EGFR proteins.PLA will be tested to recognize increased protein-protein interactionsof EGFR and interacting proteins in a panel of lung cancer cell lineswith known EGFR mutation status. This will include cell with activatingEGFR mutations (PC9, HCC827, H4006, H1975, H1650, and H820) and wildtypeEGFR (H23, A549, H1299, H460, etc) from the Lung SPORE cell line bank(total of nearly 50 NSCLC cell lines). It is expected to find higherdegrees of PLA signals in cells with activating EGFR mutations comparedto cells without EGFR mutations as these mutant EGFR proteins areconstitutively activated and poorly internalized. Also examined will beEGFR PPI PLA signals in lung cancer cells that have wildtype EGFR butnonetheless have some degree of sensitivity to EGFR TKI. This includesH322, H358, H292, and H1648 cells. Intermediate measurements of EGFR PPIusing PLA are expected here. This would reflect engagement of the EGFRprotein with downstream signaling complexes that occur throughmechanisms such as EGFR gene amplification and/or EGFR ligandstimulation.

The initial studies in human samples will measure interactions inpatient tumors with and without EGFR mutation tumor sections. For thesepilot studies, 20 cases with known EGFR mutation and 20 with wildtypeEGFR will be chosen. It is expected to find higher PLA signals in thetumors with mutations and this will provide experience and confidencewith human samples. To guide assay refinement in FFPE specimens, controltissue sections from PC9 cell xenografts will be included. Tumors fromuntreated mice and mice exposed to erlotinib will be collected theerlotinib treated mice will have EGFR inhibition and loss ofprotein-protein interactions. Thus, they serve as a control where it isexpected to see reduced PLA intensity. This will offer both positive andnegativedown-regulated control for the downstream patient tissuesamples, and also serve as a materials to optimize conditions fordifferent antibodies for immunohistochemistry (IHC). This is done evenif all the antibodies worked well by IF in cell lines.

Aim 3: Characterize Changes in EGFR Protein-Protein Interactions inResponse to EGFR Tyrosine Kinase Inhibitors in Primary Lung CancerXenograft Models and Patient Samples.

EGFR inhibitors are predicted to disrupt signaling complexes on the EGFRprotein. Protein interactions for some of these proteins can beinhibited by EGFR tyrosine kinase inhibitors (erlotinib). For example,the mass spectrometry data found that EGFR no longer bound Grb2, Mig6 orSts1 with treatment of cells with erlotinib (data not shown). Primarylung cancer xenograft systems and samples from patients before and afterEGFR inhibitor therapy will be used to measure differences in EGFRprotein-protein interactions. This could allow the development of PLAbased assays as pharmacodynamic markers of drug activity. First, primarylung cancer explant models will be used. A series of primary human NSCLCxenograft models from fresh patient tumor samples has been developed.Detailed morphological and molecular studies demonstrated that tumorsthat grew in the xenograft lines closely resemble the primary tumors,thus, the serially passaged xenografts provide a more clinicallyrelevant model than established cell lines. Of the 11 models, 5 haveKRAS mutations and one has a drug sensitive EGFR mutation. Tumors fromeach mouse will have a core biopsy before and after 7 days of erlotinib(100 mg/kg/day oral gavage). Tumors will be formalin fixed and sectionedfor PLA assays. Here it is expected to find reductions in proteins incomplex with EGFR as erlotinib would reduce the levels of tyrosinephosphorylated EGFR and disrupt binding of proteins such as Grb2 orMig6. Second, patient samples treated with EGFR TKI will be used. Theinventor just reported a pilot study of gefitinib in early stage NSCLCwhere patients received 4 weeks of gefitinib prior to having surgicalresection of lung cancer (43). This study procured pre-treatmentbiopsies (FFPE) and post-treatment biopsies (FFPE sample of surgicalspecimen) for each of 23 patients on the study. Changes in EGFR PPI willbe determined using PLA on these tumor samples. Measurements of EGFR PPIusing PLA will be analyzed from the pre-treatment sample and again onthe post-treatment sample. Here, it is expected that gefitinib willreduce interactions with EGFR.

Aim 4: Determine if EGFR Protein-Protein Interactions are Associatedwith Responses to EGFR Tyrosine Kinase Inhibitors in Human Lung CancerSamples.

The inventor proposes that quantitative measurements of EGFRprotein-protein interactions can be used as biomarkers to predictactivity of EGFR kinase inhibitors in patients with advanced lungcancer. PLA assays for EGFR protein-protein interactions in lung cancersamples will be developed and these measurements will be related toresponse to EGFR inhibitors in a group of patients with advanced lungcancer. The Moffitt Lung Cancer Program and SPORE has outstanding accessto tumor samples for these studies. The inventor has identified 822patients treated with EGFR TKI at Moffitt in the last 15 years. Of thesepatients, 193 have tissue available at Moffitt. Tissue microarrays willbe created from these samples and used for PLA based assays. Theinventor's laboratory has experience with constructing tissuemicroarrays used for multiple studies of biomarkers related to patientoutcomes (44-47). The inventor will correlate PLA biomarkers withresponse to EGFR TKI. Tumors from patients who received EGFR TKI(erlotinib or gefitinib) will be tested for PLA biomarkers and intensityrelated to (i) response measured by RECIST and (ii) disease-freesurvival and (iii) overall survival. It is expected that patients withhigher levels of PLA biomarkers to have better responses and bettersurvival with EGFR TKI. To avoid the problem of identifying a prognosticbiomarker, a cohort of patients who received cytotoxic chemotherapy astheir treatment will also be examined. Here it is expected to find nodifference if (i) PLA biomarkers are predictive of outcome for EGFR TKIand (ii) not prognostic in advanced lung cancer. The SPORE is currentlyproducing a second TMA for patients with advanced stage IV non-smallcell lung cancer who received chemotherapy at Moffitt. This includes acohort of nearly 200 cases of patients that had a prior surgicalprocedure and thus have tissue blocks with adequate tissue to producetissue arrays. One of the SPORE pathologists is currently building theseTMA and clinical data on these patients is being annotated through theSPORE infrastructure. Data elements include age, sex, race, histology,clinical and pathological stage at presentation, sites of metastaticdisease, response to therapy (by RECIST) and overall survival times.

Statistical Considerations and Approaches: Proximity ligation assay(PLA) will be used to quantify EGFR protein-protein interactions. PLA'smeasurement consists of number of foci and signal intensities for eachfocus. Histograms and boxplots will be used to visualize and examine thesampling distributions of number of foci, total intensities, and meanintensity per focus. Normality of the data will be examined using theAnderson-Darling test. Proper transformation such as log or square rootwill be explored before applying parametric tests to the data. The goalfor specific Aim 2 is to correlate each EGFR protein interaction ofinterests, quantified using PLA, to the EGFR mutation status of celllines. After proper transformation, two-sample t-test will be performedto examine whether the signal intensities differ between cell lines withand without EGFR mutations. When the assumption of normality isviolated, Mann-Whitney U tests will be applied. Limited attention hasbeen given to the potential of using PLA measurements as biomarkers inthe past, and the utility of using the number of foci as potentialbiomarkers is being explored. After examining the sampling distribution,if appropriate, it will be investigated if the number of foci differsbetween cell lines with and without EGFR mutations using Poissonregression. Over-dispersion will be examined. With a maximum of 6proposed interactions in this work, final number of tests (i.e., totalnumber of interactions) will be determined by assay development forAim 1. The same analysis procedure will be carried out for the datacollected from clinical samples. The goal for specific Aim 3 is toassess if there is a difference for each protein interaction, quantifiedby PLA, before and after EGFR TKI (erloitnib) treatment. The Pre- andpost-treatment samples will be taken from the same xenograph mouse.Paired t-tests or Wilcoxon signed-rank tests will be performed toexamine the difference of the signal intensities between pre- andpost-treatment for each EGFR protein interaction. The same analysisprocedure will be performed for the clinical samples. The goal forspecific Aim 4 is to identify if each of the EGFR protein-proteininteractions is a predictive factor to EGFR tyrosine kinase inhibitors(TKI) but not a general prognostic factor (following the definitionsprovided by Clark(48, 49). Briefly, a predictive factor has adifferential benefit from the TKI treatment that depends on the statusof the predictive biomarker while a prognostic factor is thought of as ameasure of the natural history of the disease. In statistical terms, apredictive factor could be identified when the interaction term betweentreatment group and biomarker status in the statistical model issignificant. The EGFR protein-protein interactions will be quantifiedfor ˜193 human lung cancer samples from patients with advanced stage andabout same amount of samples from patients treated with chemotherapyusing the tissue microarray arrays (TMAs). The inventor will firstexamine if there is any spatial heterogeneity visually using heat map aswell as analytically using regression models (50, 51). Each EGFRprotein-protein interaction will be classified as high- orlow-expression group by searching the optimal cut point using themaximal logrank method with adjusted P-values for multiple looks, theassociation between the overall survival of patients and high- andlow-expression groups will be assessed using the Kaplan Meier curve andlog-rank test. Gender, histology, and smoking status will be includedalong with the treatment group (EGFR TKI vs. chemotherapy) and biomarkerstatus (high vs. low) in the multivariable Cox regression model. Theinteraction term between treatment group and biomarker status will betested to determine if the EGFR protein-protein interaction is apredictive marker. Stratified analyses will also be performed andfacilitate the visualization of the differential clinical benefit forthe patients in different biomarker group using Kaplan Meier curve. Foreach pair of EGFR protein-protein interaction, one Cox multivariableregression model will first be performed. The assumption of proportionalhazards will be evaluated by examining residual plots and by includingtime-dependent terms in the models. Similar analyses will be performedfor the disease free survival. After proper transforming the signalintensity for each EGFR protein-protein interaction, one-way ANOVA willbe performed to examine the intensity difference among differentresponse groups (CR/PR, SD, and PD). When parametric assumptions areviolated, Kruskal-Wallis test will be performed. The overall goal is todevelop and evaluate the proximity ligation assays for each pair ofprotein interactions to examine if they are effective biomarkers.Multiple hypothesis testing will be addressed using 10% false discoveryrate to declare statistical significance. Limited preliminary data arecurrently available, and no statistical power is calculated for samplesize justification. The experiments proposed in this work will give usbetter estimate of the effective size and sample size for future work.To examine if each protein-protein interaction is a good biomarker toclassify disease control (CR/PR/SD) from disease progression (PD), theinventor will evaluate the sensitivity and specificity. ROC analysiswill be performed to evaluate the area under the curve (AUC) consideringdifferent thresholds. Combination of several markers may lead to a moreaccurate classification, all selected and quantified biomarkers will beincluded and examined using several multivariable models such aslogistic regression and methods with built-in variable selectionprocedures such as Hybrid huberized support vector machines (HHSVM) andBayesian variable selection approach (52, 53).

The rich MS data that has been experimentally generated for proteinpairs can be capitalized upon to evaluate and test for interaction.Numerous papers have been published on PLA (23, 25-27, 54). Smallinterfering RNA (siRNA) molecules can be used to eliminate proteins inPLA assays as another method to develop specific assays (55). Theinventor expects that siRNA treated cells will show reduced or absentsignaling from PLA assays demonstrating specificity of the assay againstthe interaction studied. The inventor has substantial experience withAQUA; HistoRx will provide additional expertise and advice, such asproviding lists of validated antibodies and help with troubleshooting(46, 47, 56-58).

The SPORE and Moffitt Total Cancer Care initiative have standardoperating protocols to minimize these variables. Spatial heterogeneityin interactions measured by PLA could be observed across a tumor. Coreto core variation can be examined as it is typical to have 3 cores pertumor for the tissue microarrays. Whole sections of tumors can also beexamined to determine spatial variability of PLA signals within the sametumor. The PLA signals may be relatively weak compared to those inconventional AQUA targets detected with IF. However, one advantage ofthe new version of AQUA is better dynamic range that can distinguishthose with higher and lower in PPI signal detected with PLA even with alower cutoff. Finally, most of the interactions being examined are basedon phosphorylation dependent interactions (for example, phosphotyrosinemotifs interacting with Src homology-2 (SH2) domains). Thus, somesignals could be weak if samples are not prepared well andphosphorylation maintained. The ability exists to examine other signalsthat are likely more stable based on the MS data (FIG. 1); this includesHsp90:EGFR, Hsp90:Cdc37, and other binary interactions.

FIGS. 3A-F show results of an EGFR-GRB2 PLA in situ. FIGS. 4A-B showresults of EGFR-Grb2 PLA in the PC9 lung cancer cell line. FIGS. 5A-Bshow results of EGFR-Grb2 PLA in lung cancer tumor tissues (in vivo).FIGS. 6A-B show results of ALK-Grb2 PLA in H3122 cells. FIGS. 7 and 8show, respectively, results of EGFR:Grb2 PLA in cell lines related toEGFR mutation status, and results of EGFR:Grb2 in the 21 tissuemicroarray related to EGFR and KRAS mutation.

Example 2 Duolink to Test EGFR and Grb2 Interaction Reagent Information:Antibody:

rGRb2 (Rabbit polyclone: C23, SC-255 Santa Cruz),

mEGFR (mouse monoclonal antibody, LS-C88071/21583, LIFESPANBiosciences,)

Working dilutions: m-EGFR (1:250)+r-GRb2 (1:100)

-   -   Each well is one square cm, so at least 44 ul/well of PLA        solution is needed. But 100 ul for antibodies and 250 ul for        washing solutions are applied.

Experimental Procedure:

-   1. Plate approximately 3×10⁴ cells in 350□l media per well of a    Lab-Tek II 8-well chamber slide. Grow cells for ˜36 hours.-   2. Aspirate media and add 250 μl 4% paraformaldehyde in PBS per well    with a subsequent incubation for 20 minutes at 4°.-   3. Aspirate the paraformaldehyde, then go to step 4.-   4. 250 μl 0.5% Triton-X 100 per well from a 10% stock diluted in PBS    with a subsequent incubation for 10 minutes at room temperature with    gently rocking. Aspirate the Triton-X 100, wash each well once with    250 μl PBS, and add 250βl 1.5% BSA in PBS (from 10× stock) per well    for blocking with gentle rocking for 30 minutes at room temperature.-   5. Add the appropriate primary antibodies [m-EGFR (1:250)+r-GRb2    (1:100)] in PBS and incubate at room temperature for 2 hours with    gentle rocking.-   6. Aspirate the primary antibody solution and wash each well    briefly, 3× with 250 μl of 1×PBS.

7. Secondary antibody-PLA probes: Mix and dilute the two PLA probes 1:5in PBS (9 minus+9 μl plus+27 μl PBS=45/well but 44/well, (otherwise thewells could not be covered), incubate the slides in a pre-heatedhumidity chamber for 1 hour at 37 C.

-   8. Ligation: Dilute the Ligation stock 1:5 in MQW while waiting (9    μl minus+36 μl MQW=45). Aspirate the PLA probes and wash 2×2 minutes    in PBS rocking. Then, add Ligase into ligation solution 1:40 then    add 44 μl/well, immediately incubate for 30 minutes at 37 C.-   9. Amplification-hybridations-fluorecences probes: Dilute the    Amplification stock 1:5 in MQW while waiting (9 μl minus+36 μl    MQW=45). Aspirate the Ligation solution and wash 2×2 minutes in PBS    rocking, then add Polymerase 1:80 into amplification solution, add    polymerase-amplification solution 44 μl/well, incubate for 100    minutes at 37 degrees C. (avoid light).-   10. Aspirate the amplification solution wash with 1× Wash Buffer B    for 2×10 minutes Wash the slide in 0.1× Wash Buffer B. Let the    slides dry at RT in the dark. Mount the slides with Duolink II    Mounting Medium. Dry it for overnight (avoid light throughout the    step).-   11. Observe under confocal microscopy.

Example 3 Duolink to test EGFR and Grb2 Interaction in Formalin-FixedParaffin-Embedded (FFPE) Tissue/Cell Pellets Reagent Information:

Antibodies for FFPE tissues and FFPE cell pellets:mEGFR (LIFESPAN Bioscie, LS-C88071/21583), 1:50rGrb2 (Santa Cruz polyclone C23, SC-255), 1:50

Experimental Procedure:

-   1. Cut slides in 3 um using window type transfer system and adhesive    coated slides (Instrumedics). After UV exposure for one minute, peel    the tape in TPC solvent, than hydration of tissue/cells section.-   2. Put slides in PT4 media and heat with microwave for ten minutes,    and then cool down for thirty minutes at room temperature.-   3. Add 250 μl 0.5% Triton-X 100 per well from a 10% stock diluted in    PBS with a subsequent incubation for 10 minutes at room temperature    with gently rocking. Aspirate the Triton-X 100, wash each well once    with 250 μl PBS, and add 250 μl 1.5% BSA in PBS (from 10× stock) per    well for blocking with gentle rocking for thirty minutes at room    temperature.-   4. Add the appropriate primary antibodies at the pre-determined    optimal concentration (see the layout above) in PBS and incubate at    room temperature for over night at 4 C with gentle rocking.-   5. Aspirate the primary antibody solution and wash the slides    briefly three times with 250 μl in PBS.-   6. Prepare and incubate secondary antibody-PLA probes: Mix and    dilute the two PLA probes 1:5 in PBS (9 μl minus+9 μl plus+27 μl    PBS=45 well but 44/squar cm to cover the whole section), add 88 μl    Mixed PLA probes well, incubate the slides in a pre-heated humidity    chamber for one hour at 37 degrees C.-   7. Ligation: Dilute the Ligation stock 1:5 in MQW while waiting (9    μl minus+36 μl MQW=45 μl). Aspirate the PLA probes and wash two    times, each for 2 minutes in PBS with gently rocking, Then, add    ligase into ligation solution 1:40 then add 44 μl/section,    immediately incubate for thirty minutes at 37 degrees C.-   8. Prepare and incubate the amplification-hybridization-fluorescence    probes: Dilute the amplification stock 1:5 in MQW while waiting (9    μl minus+36 MQW=45 μl). Aspirate the Ligation solution and wash two    minutes in PBS rocking two times, then add polymerase 1:80 into    amplification solution, add polymerase-amplification solution 44    μl/well, incubate for 100 minutes at 37 degrees C. (avoid light).-   9. Aspirate the Amplification solution, wash with 1× Wash Buffer B    for 10 minutes for 2 times. Pip the slides in 0.1× Wash Buffer B    (Duolink). Then WGA (this step can be omitted) for five minutes    (avoid light).-   10. Wash with PBS five minutes for three times. Then let the slides    dry at room temperature in a dark chamber for twenty minutes. Mount    the dried slides with Mounting Medium. Dry the slides for overnight    before observation with confocal microscopy.

Example 4 Protein-Protein Interactions for all Human Tyrosine Kinases

Bioinformatic tools were used to identify all reported protein-proteininteractions for all human tyrosine kinases (2,848 interactions).

-   -   1) 90 of TK (tyrosine kinase) protein were queried from        Kinase.com and verified and annotated with gene symbol.        Kinase.com is produced by at the Salk Institute and has the full        complement of protein kinases in any sequenced genome, including        extensive KinBase database, papers and supporting material from        Sugen and the Salk Institute.    -   2) The list of the TK gene symbol was queried with MiMI        (Michigan Molecular Interactions) plug from Cytoscape, an        open-source software platform for visualizing complex networks        and integrating these with any type of attribute data. The        network data were save as an Excel file. The NCIBI MiMI is part        of the NIH's National Center for Integrative Biomedical        Informatics (NCIBI), which provides access to the knowledge and        data merged and integrated from numerous protein interactions        databases.    -   3) 90 TK proteins have been reported for interaction with 1,104        proteins, and 2,848 interactions have been reported. The        interactions were verified via the following databases: BIND,        CCSB, DIP, GRID, HPRD, IntAct, MDC, MINT, reactome and PubMed.        The interaction network was rebuilt with Cytoscape.

Some of the resulting data is presented in Tables 3-5. Tables 3 and 4list ninety human tyrosine kinases, with aliases and accession numbers.Table 5 lists human tyrosine kinase interactions (node 1=tyrosinekinase; node 2=interacting protein).

TABLE 3 Human Tyrosine Kinases (with aliases) Gene ♦ Species↑Classification♦ Other Names ABL1 Human TK:Abl: c-ab1, c-ABL, JTK7, p150,ABL1, ABL, v-abl ABL2 Human TK:Abl: ABLL, ARG, ABL2, ABLL ACK HumanTK:Ack: ACK1, TNK2, ACK, ACK1, FLJ44758, FLJ45547, p21cdc42Hs ALK HumanTK:ALK: ALK, CD246, TFG/ALK AXL Human TK:Axl: UFO, Ark, AXL, UFO BLKHuman TK:Src:SrcB MGC10442, BLK, MGC10442 BMX Human TK:Tec: PSCTK3,PSCTK2, ETK, BMX, PSCTK3 BRK Human TK:Src:SRM PTK6, PTK6, BRK, FLJ42088BTK Human TK:Tec: XLA, PSCTK1, IMD1, BPK, ATK, AGMX1, AT, BTK,MGC126261, MGC126262, XLA CCK4 Human TK:CCK4: PTK7, PTK7, CCK4 CSK HumanTK:Csk: CYL, CSK, MGC117393 CTK Human TK:Csk: MGC2101, MGC1708, Lsk,HYL, HHYLTK, DKFZp434N1212, CHK, MATK, CTK, HYLTK, MGC2101 DDR1 HumanTK:DDR: trkE, TRKE, RTK6, PTK3A, PTK3, NTRK4, MCK10, EDDR1, DDR, NEP,CAK, DDR1, CD167 DDR2 Human TK:DDR: TYRO10, NTRKR3, TKT, DDR2, MIG20a,TYRO10 EGFR Human TK:EGFR: ERBB1, ERBB, EGFRvIII, EGFR, ERBB1, mENAEphA1 Human TK:Eph: EPHT1, EPHT, EPH, EPHA1, EPHT1, MGC163163 EphA10Human TK:Eph: EPHA10, FLJ16103, FLJ33655, MGC43817 EphA2 Human TK:Eph:ECK, EPHA2, ECK EphA3 Human TK:Eph: HEK4, HEK, ETK1, TYRO4, ETK, EPHA3,HEK4 EphA4 Human TK:Eph: TYRO1, HEK8, EPHA4, SEK, TYRO1 EphA5 HumanTK:Eph: Hs.31092, Hs.194771, HEK7, EPHA5, CEK7, EHK1, TYRO4 EphA6 HumanTK:Eph: EPHA6, DKFZp434C1418, EPA6, FLJ35246, PRO57066 EphA7 HumanTK:Eph: HEK11, EPHA7, EHK3, HEK11 EphA8 Human TK:Eph: KIAA1459, HEK3,EEK, EPHA8, KIAA1459 EphB1 Human TK:Eph: Hek6, HEK6, EPHT2, ELK, NET,EPHB1, FLJ37986 EphB2 Human TK:Eph: Tyro5, Hek5, HEK5, EPHT3, DRT, ERK,EPHB2, CAPB, MGC87492, PCBC, Tyro5 EphB3 Human TK:Eph: TYRO6, HEK2,ETK2, EPHB3, TYRO6 EphB4 Human TK:Eph: TYRO11, MYK1, HTK, EPHB4, TYRO11EphB6 Human TK:Eph: HEP, EPHB6, HEP, MGC129910, MGC129911 ErbB2 HumanTK:EGFR: c-erbB2, c-erbB-2, TKR1, NGL, Hs.323910, Hs.103992, HER2,HER-2, NEU, ERBB2, HER-2/neu, c-erb B2 ErbB3 Human TK:EGFR: Hs.199067,Hs.167387, Hs.167386, HER3, ERBB3, ErbB-3, MDA-BF-1, MGC88033, c-erbB-3,c-erbB3, erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3 ErbB4 HumanTK:EGFR: HER4, ERBB4, HER4, MGC138404, p180erbB4 FAK Human TK:FAK:pp125FAK, FAKpp125, FAK1, FADK, PTK2, FAK, pp125FAK FER Human TK:Fer:TYK3, NCP94, FER, TYK3 FES Human TK:Fer: FPS, FES, FPS FGFR1 HumanTK:FGFR: LOC51033, N-SAM, FLT2, FLJ14326, CEK, C-FGR, BFGFR, H5, H4, H3,H2, FLG, FGFR1, CD331, FGFBR, HBGFR, KAL2, N-SAM FGFR2 Human TK:FGFR:TK25, TK14, KGFR, K-SAM, JWS, ECT1, CFD1, CEK3, BFR-1, BEK, FGFR2,CD332, TK25 FGFR3 Human TK:FGFR: FGFR3, ACH, CD333, CEK2, HSFGFR3EX,JTK4 FGFR4 Human TK:FGFR: TKF, JTK2, FGFR4, CD334, MGC20292, TKF FGRHuman TK:Src:SrcA SRC2, FGR, FLJ43153, MGC75096, SRC2, c-fgr, c-src2,p55c-fgr, p58c-fgr FLT1 Human TK:VEGFR: VEGFR1, VEGFR-1, FLT-1, FLT,FLT1, VEGFR1 FLT3 Human TK:PDGFR: FLK2, CD135, STK1, FLT3, FLK2 FLT4Human TK:VEGFR: VEGFR3, PCL, FLT4, FLT41, VEGFR3 FMS Human TK:PDGFR:CSF-1R, CD115, CSF1R, C-FMS, CSFR, FIM2, FMS FRK Human TK:Src:Frk RAK,GTK, FRK, PTK5, RAK FYN Human TK:Src:SrcA MGC45350, SYN, SLK, FYN,MGC45350 HCK Human TK:Src:SrcB p59hck, p56hck, JTK9, HCK IGF1R HumanTK:InsR: JTK13, IGF1R, CD221, IGFIR, JTK13, MGC142170, MGC142172,MGC18216 INSR Human TK:InsR: INSR, CD220, HHF5 IRR Human TK:InsR: INSRR,INSRR, IRR ITK Human TK:Tec: LYK, PSCTK2, EMT, ITK, LYK, MGC126257,MGC126258 JAK1 Human TK:Jak: JAK1A, JAK1, JAK1A, JAK1B JAK2 HumanTK:Jak: JAK2 JAK3 Human TK:Jak: LJAK, L-JAK, JAKL, JAK3, JAK-3,JAK3_HUMAN, LJAK KDR Human TK:VEGFR: VEGFR2, VEGFR-2, VEGFR, Hs.KDR,Hs.12337, FLK1, FLK-1, KDR, CD309, VEGFR2 KIT Human TK:PDGFR: SCFR,CD117, PBT, KIT, C-Kit, SCFR LCK Human TK:Src:SrcB p56lck, LCK, YT16,p56lck, pp58lck LMR1 Human TK:Lmr: KIAA0641, AATYK, AATK, KIAA0641,LMR1, LMTK1 LMR2 Human TK:Lmr: KIAA1079, LMTK2, AATYK2, BREK, KIAA1079,KPI- 2, KPI2, LMR2, cprk LMR3 Human TK:Lmr: LMTK3, KIAA1883, LMR3,TYKLM3 LTK Human TK:ALK: TYK1, LTK, TYK1 LYN Human TK:Src:SrcB JTK8,LYN, FLJ26625, JTK8 MER Human TK:Axl: mer, c-mer, C-MER, MERTK, MER,MGC133349 MET Human TK:Met: HGFR, C-MET, RCCP2, MET, HGFR MUSK HumanTK:Musk: MUSK, MGC126323, MGC126324 PDGFRa Human TK:PDGFR: PDGFR2,CD140A, PDGFRA, MGC74795, PDGFR2, Rhe-PDGFRA PDGFRb Human TK:PDGFR:PDGFR1, PDGFR, PDGF-R-beta, JTK12, CD140B, PDGFRB, PDGFR1 PYK2 HumanTK:FAK: PTK2B, PKB, CADTK, CAKB, CAK_beta, FAK2, PTK, RAFTK, FADK2,PYK2, RAFTK RET Human TK:Ret: PTC, CDHF12, HSCR1, Hs.168114, Hs.RET,MEN2A, MEN2B, MTC1, RET51, RET, RET-ELE1, RET51 RON Human TK:Met: MST1R,CDw136, CDw136, PTK8, RON ROR1 Human TK:Ror: NTRKR1, dJ537F10.1, ROR1,MGC99659, dJ537F10.1 ROR2 Human TK:Ror: BDB, BDB1, NTRKR2, ROR2,MGC163394, NTRKR2 ROS Human TK:Sev: ROS1, MCF3, MCF3, ROS RYK HumanTK:Ryk: D3S3195, RYK1, RYK, JTK5, JTK5A, RYK1 SRC Human TK:Src:SrcASRC1, ASV, SRC, ASV, c-SRC, p60-Src SRM Human TK:Src:SRM SRMS,C20orf148, SRM, dJ697K14.1 SuRTK106 Human TK:TK-Unique: DKFZp761P1010,DKFZP761P1010, STYK1, NOK, SuRTK106 SYK Human TK:Syk: SYK TEC HumanTK:Tec: PSCTK4, TEC, MGC126760, MGC126762, PSCTK4 TIE1 Human TK:Tie:TIE, JTK14, TIE1, JTK14 TIE2 Human TK:Tie: VMCM1, TEK, HPK-6, TIE-2,VMCM, CD202B, TIE2, VMCM1 TNK1 Human TK:Ack: TNK1, MGC46193 TRKA HumanTK:Trk: NTRK1, TRK, MTC, TRK-A, DKFZp781I14186, TRK1, TRKA, p140-TrkATRKB Human TK:Trk: NTRK2, NTRK2, GP145-TrkB, TRKB TRKC Human TK:Trk:NTRK3, NTRK3, TRKC, gp145(trkC) TXK Human TK:Tec: BTKL, PSCTK5, RLK,TKL, TXK, MGC22473, PTK4, TKL TYK2 Human TK:Jak: JTK1, TYK2, JTK1 TYRO3Human TK:Axl: DTK, RSE, SKY, TIF, TYRO3, BYK, Brt, Tif YES HumanTK:Src:SrcA YES1, C-YES, P61-YES, c-yes, HsT441, Yes ZAP70 Human TK:Syk:STD, SRK, ZAP-70, ZAP70, TZK, ZAP-70

TABLE 4 Human Tyrosine Kinases (with accession numbers) Gene_SymbolProbeset Accession Gene_ID ABL1 202123_s_at NM_005157 NM_007313 25 ABL2231907_at 206411_s_at 226893_at NM_005158 NM_001168237 27 NM_001168239NM_007314 NM_001136000 NM_001168236 NM_001168238 TNK2 203839_s_at216439_at AK225786 NM_001010938 NM_005781 10188 1555557_a_at 228279_s_atAB209338 203838_s_at ALK 208211_s_at 208212_s_at NM_004304 238 AXL202685_s_at 202686_s_at NM_001699 NM_021913 558 BLK 244394_at 236820_at206255_at NM_001715 NG_023543 BC038555 640 210934_at BMX 206464_at242967_at NM_203281 NG_013227 NM_001721 660 PTK6 1553114_a_at NM_0059755753 BTK 205504_at NM_000061 695 PTK7 1555324_at 207011_s_at BC046109NM_002821 NM_152881 5754 NM_152880 NM_152882 CSK 202329_at NM_001127190NM_004383 1445 MATK 206267_s_at NM_002378 NM_139354 NM_139355 4145 DDR1208779_x_at 207169_x_at NM_001202523 NM_001202521 780 210749_x_at1007_s_at NM_013993 NM_001202522 NM_001954 NM_013994 DDR2 227561_at205168_at NM_001014796 NM_006182 AY423733 4921 EGFR 211550_at210984_x_at 211551_at NM_201284 NM_201282 NM_201283 1956 211607_x_at201984_s_at NM_005228 K03193 201983_s_at 1565483_at 1565484_x_at EPHA1215804_at 205977_s_at NM_005232 EU826604 2041 EPHA10 1553371_at236073_at NM_001099439 NM_173641 284656 EPHA2 203499_at NM_004431 1969EPHA3 206071_s_at 206070_s_at NM_182644 NM_005233 2042 211164_at EPHA4227449_at 228948_at 229374_at NM_004438 2043 206114_at EPHA5 215664_s_at216837_at 237939_at L36644 NM_004439 NM_182472 2044 EPHA6 233184_at1561396_at NM_173655 NM_001080448 285220 EPHA7 1554629_at 229288_at238533_at NM_004440 BC027940 2045 206852_at EPHA8 231796_at 1554069_atNM_020526 NM_001006943 2046 EPHB1 230425_at 211898_s_at NM_004441 2047210753_s_at EPHB2 209588_at 209589_s_at NM_017449 NM_004442 2048211165_x_at 210651_s_at EPHB3 204600_at 1438_at NM_004443 2049 EPHB4202894_at 216680_s_at NM_004444 2050 EPHB6 204718_at NM_004445 2051ERBB2 210930_s_at 216836_s_at NM_001005862 NM_004448 2064 ERBB31563252_at 202454_s_at NM_001982 U88360 2065 226213_at 1563253_s_atERBB4 206794_at 241581_at 233494_at NM_001042599 AC108220 NM_005235 2066214053_at 233498_at AK024204 PTK2 1559529_at 208820_at NM_153831NM_005607 AC105009 5747 207821_s_at 241453_at BC043202 NM_001199649 FER227579_at 206412_at AC116428 NM_005246 2241 FES 205418_at NM_001143783NM_001143785 2242 NM_002005 NM_001143784 FGFR1 215404_x_at 207822_at226705_at AK024388 NM_023106 NM_015850 M34187 2260 211535_s_at210973_s_at NM_023105 NM_001174067 207937_x_at NM_001174066 NM_001174065NM_023110 NM_001174063 NM_001174064 FGFR2 211399_at 203639_s_at208225_at NM_001144915 NM_001144918 AB030077 2263 208234_x_at 211398_atNM_001144917 AB030075 M87772 211401_s_at 208228_s_at NM_001144916NM_001144913 203638_s_at 211400_at NM_022970 NM_001144914 AB030073NM_001144919 NM_000141 FGFR3 204380_s_at 204379_s_at NM_000142NM_001163213 NM_022965 2261 FGFR4 1554961_at 211237_s_at NM_002011NM_022963 AF359241 2264 204579_at 1554962_a_at NM_213647 FGR 208438_s_atNM_001042729 NM_005248 2268 NM_001042747 FLT1 210287_s_at 226497_s_atNM_001159920 NM_002019 2321 222033_s_at 226498_at FLT3 206674_atNM_004119 2322 FLT4 234379_at 210316_at 229902_at NM_002020 NM_1829252324 CSF1R 203104_at NM_005211 1436 FRK 207178_s_at NM_002031 2444 FYN217697_at 210105_s_at NM_002037 NM_153048 AL109916 2534 216033_s_atNM_153047 HCK 208018_s_at NM_001172129 NM_001172131 3055 NM_002110NM_001172130 NM_001172133 NM_001172132 IGF1R 208441_at 243358_at203627_at AF020763 AC055807 NM_000875 3480 225330_at 238544_at 237377_at237881_at 203628_at INSR 227432_s_at 226450_at NM_000208 AB208861AC010526 3643 213792_s_at 243002_at NM_001079817 226212_s_at 226216_at207851_s_at INSRR 215776_at NM_014215 3645 ITK 211339_s_at NM_0055463702 JAK1 1552611_a_at 239695_at NM_002227 BX648044 3716 1552610_a_at201648_at JAK2 205842_s_at 205841_at NM_004972 BC043187 3717 1562031_atJAK3 211109_at 211108_s_at 207187_at NM_000215 U31601 3718 227677_at KDR203934_at NM_002253 3791 KIT 205051_s_at NM_000222 NM_001093772 3815 LCK204891_s_at 204890_s_at NM_001042771 NM_005356 3932 AATK 205986_atXR_115154 NM_001080395 XR_111752 9625 LMTK2 235307_at 206223_at226375_at NM_014916 NG_013375 22853 LMTK3 1557103_a_at NM_001080434114783 LTK 207106_s_at 217184_s_at NM_002344 NM_206961 NM_001135685 4058LYN 202625_at 210754_s_at NM_001111097 NM_002350 4067 202626_s_at MERTK211913_s_at 206028_s_at NM_006343 10461 MET 213807_x_at 203510_atNM_001127500 NM_000245 4233 211599_x_at 213816_s_at MUSK 207632_at207633_s_at NM_001166281 NM_001166280 4593 241122_s_at NM_005592 PDGFRA211533_at 237696_at 1554828_at L25829 AC098587 M22734 BC015186 5156203131_at 215305_at NM_006206 PDGFRB 202273_at NM_002609 5159 PTK2B203110_at 203111_s_at NM_004103 NM_173175 NM_173174 2185 NM_173176 RET215771_x_at 211421_s_at NM_020630 NM_020975 5979 205879_x_at MST1R205455_at NM_002447 4486 ROR1 205805_s_at 211057_at NM_001083592NM_005012 4919 ROR2 231000_at 205578_at NM_004560 BC051273 4920 ROS1207569_at NM_002944 6098 RYK 214172_x_at 202853_s_at AC107310NM_001005861 NM_002958 6259 216976_s_at 238210_at SRC 1565082_x_at237103_at NG_023033 NM_198291 NM_005417 6714 213324_at 1565080_at221284_s_at 1558211_s_at SRM 201516_at NM_003132 6723 STYK1 221696_s_at220030_at NM_018423 55359 SYK 226068_at 209269_s_at BX647192NM_001135052 NM_001174168 6850 207540_s_at 244023_at NM_001174167NM_003177 TEC 206301_at NM_003215 7006 TIE1 1560657_at 204468_s_atNM_005424 AL833389 7075 TEK 217711_at 206702_at NM_000459 7010 TNK1217149_x_at 205793_x_at NM_003985 8711 NTRK1 208605_s_at NM_001007792NM_001012331 4914 NM_002529 NTRK2 214680_at 207152_at 236095_atNM_006180 NM_001018065 4915 221796_at 229463_at 221795_at NM_001007097NM_001018064 BX649001 NM_001018066 NTRK3 217033_x_at 217377_x_atNM_002530 NM_001012338 4916 215115_x_at 215025_at NM_001007156206462_s_at 1557795_s_at 228849_at TXK 206828_at NM_003328 7294 TYK2205546_s_at NM_003331 7297 TYRO3 211432_s_at 211431_s_at NM_006293X72886 7301 1566934_at YES1 202932_at 202933_s_at NM_005433 7525 ZAP701555613_a_at 214032_at NM_207519 NM_001079 7535

TABLE 5 Human Tyrosine Kinase Interactions (node 1 = tyrosine kinase;node 2 = interacting protein) Node1 Node2 int_DataBase AATK STK39 (GRID)ABL1 ABI1 (BIND; GRID; HPRD) ABL1 ABI2 (GRID; HPRD; MINT) ABL1 ABL2(BIND; GRID; HPRD) ABL1 ACTA1 (HPRD) ABL1 ADAM15 (GRID; HPRD) ABL1 APBB1(HPRD) ABL1 APP (HPRD) ABL1 ATM (DIP; GRID; HPRD; IntAct) ABL1 ATR(GRID) ABL1 BCAR1 (GRID; HPRD) ABL1 BCL2L1 (MINT) ABL1 BCR (BIND; GRID;HPRD) ABL1 BIN1 (HPRD) ABL1 BRCA1 (GRID; HPRD) ABL1 BTK (HPRD) ABL1 C3(GRID; HPRD) ABL1 CABLES1 (HPRD) ABL1 CABLES2 (GRID; HPRD) ABL1 CASP9(HPRD) ABL1 CAV1 (HPRD) ABL1 CBL (GRID; HPRD; MINT) ABL1 CD19 (HPRD)ABL1 CDC2 (HPRD) ABL1 CDK5 (HPRD) ABL1 CDKN1B (HPRD) ABL1 CREB1 (GRID)ABL1 CRK (BIND; HPRD) ABL1 CRKL (GRID; HPRD) ABL1 CTNND2 (GRID; HPRD)ABL1 DDB1 (HPRD) ABL1 DDB2 (HPRD) ABL1 DOK1 (HPRD) ABL1 DOK3 (HPRD) ABL1EGFR (MINT) ABL1 EP300 (MINT) ABL1 EPHB2 (GRID; HPRD) ABL1 ERBB2 (MINT)ABL1 ERBB3 (MINT) ABL1 ERBB4 (MINT) ABL1 EVL (GRID; HPRD) ABL1 GPX1(GRID; HPRD) ABL1 GRB10 (GRID) ABL1 GRB2 (GRID; HPRD) ABL1 GRIN2D (HPRD;MINT) ABL1 HCK (GRID; HPRD) ABL1 INPPL1 (GRID; HPRD) ABL1 JAK1 (GRID;HPRD) ABL1 JAK2 (HPRD) ABL1 JUN (HPRD) ABL1 KIT (HPRD) ABL1 MAP4K1(HPRD) ABL1 MAP4K5 (GRID; HPRD) ABL1 MAPT (HPRD) ABL1 MDM2 (GRID; HPRD)ABL1 MUC1 (HPRD; MINT) ABL1 NCF1 (BIND) ABL1 NCK1 (GRID; HPRD) ABL1NCSTN (GRID; HPRD) ABL1 NEDD9 (GRID; HPRD) ABL1 NTRK1 (GRID; HPRD) ABL1PAG1 (GRID; HPRD) ABL1 PAK2 (GRID; HPRD) ABL1 PDE4D (BIND) ABL1 PIK3R1(BIND; GRID; HPRD) ABL1 PLCG1 (BIND; HPRD) ABL1 PLSCR1 (HPRD; MINT) ABL1POLR2A (HPRD) ABL1 PRKCD (MINT) ABL1 PRKD1 (HPRD) ABL1 PRKDC (GRID;HPRD; MINT) ABL1 PSTPIP1 (GRID; HPRD) ABL1 PTPN12 (HPRD) ABL1 PTPN18(HPRD) ABL1 PTPN6 (GRID; HPRD) ABL1 PXN (GRID; HPRD) ABL1 RAD51 (GRID;HPRD; MINT) ABL1 RAD52 (HPRD) ABL1 RAD9A (GRID; HPRD) ABL1 RAN (GRID;HPRD) ABL1 RASA1 (BIND; HPRD) ABL1 RB1 (BIND; GRID; HPRD; MINT) ABL1RFX1 (GRID; HPRD) ABL1 RIN1 (BIND; GRID; HPRD; IntAct; MINT) ABL1 ROBO1(BIND; HPRD) ABL1 ROS1 (GRID; HPRD) ABL1 RYBP (GRID) ABL1 SFN (HPRD;IntAct) ABL1 SH3BP1 (GRID) ABL1 SH3BP2 (HPRD) ABL1 SHD (GRID; HPRD) ABL1SHE (HPRD) ABL1 SLC9A2 (GRID; HPRD) ABL1 SORBS1 (GRID; HPRD) ABL1 SORBS2(GRID; HPRD) ABL1 SOS2 (HPRD) ABL1 SRC (BIND; HPRD) ABL1 ST5 (GRID;HPRD) ABL1 TERT (HPRD; MINT) ABL1 TP53 (GRID; HPRD; MINT) ABL1 TP73(GRID; HPRD; MINT) ABL1 TRAF6 (HPRD) ABL1 TUB (GRID; HPRD) ABL1 WASF1(BIND; HPRD; MINT) ABL1 WASL (HPRD) ABL1 XPO1 (BIND) ABL1 XRCC6 (GRID;HPRD) ABL1 YTHDC1 (GRID; HPRD) ABL1 YWHAB (HPRD) ABL1 YWHAE (HPRD) ABL1YWHAG (HPRD) ABL1 YWHAH (BIND; HPRD) ABL1 YWHAZ (HPRD) ABL1 ZAP70 (GRID;HPRD) ABL1 ZDHHC16 (GRID; HPRD) ABL2 ABI2 (BIND) ABL2 BCR (BIND; HPRD)ABL2 CAT (GRID; HPRD) ABL2 CRK (BIND; GRID; HPRD; IntAct) ABL2 EGFR(MINT) ABL2 EPHB2 (HPRD) ABL2 ERBB2 (MINT) ABL2 ERBB3 (MINT) ABL2 ERBB4(MINT) ABL2 GPX1 (HPRD) ABL2 HCK (GRID; HPRD) ABL2 HRAS (MINT) ABL2 JAK1(GRID) ABL2 ONECUT1 (BIND) ABL2 RIN1 (BIND; DIP; IntAct; MINT) ABL2SIVA1 (HPRD) ABL2 SORBS2 (GRID; HPRD) ALK CENPF (IntAct) ALK EIF4B(IntAct) ALK EPHA1 (IntAct) ALK EPHB3 (IntAct) ALK HSP90AA1 (GRID) ALKHSPD1 (IntAct) ALK IRS1 (GRID; HPRD; IntAct) ALK JAK3 (GRID; HPRD) ALKMAP3K1 (IntAct) ALK MAP3K3 (IntAct; MINT) ALK MDK (HPRD) ALK NPM1 (HPRD)ALK PLCG1 (GRID; HPRD) ALK PTN (DIP; GRID; HPRD) ALK PTPRZ1 (HPRD) ALKRASA1 (GRID) ALK SHC1 (GRID; HPRD) ALK SHC3 (GRID; HPRD) ALK SOCS5(IntAct) ALK STAT3 (IntAct) ALK TNFRSF8 (HPRD) ALK ZC3HC1 (HPRD) AXLADAM10 (HPRD) AXL CBL (HPRD) AXL CSK (BIND) AXL GAS6 (GRID; HPRD; MINT)AXL GRB2 (BIND; CCSB; GRID; HPRD) AXL IL15RA (HPRD) AXL IL2RG (HPRD) AXLLCK (BIND; CCSB; GRID; HPRD) AXL NCK2 (BIND; HPRD) AXL PIK3R1 (BIND;HPRD) AXL PIK3R2 (BIND; GRID; HPRD) AXL PIK3R3 (BIND; HPRD) AXL PLCG1(BIND; HPRD) AXL PTPN11 (HPRD) AXL RANBP9 (BIND; HPRD) AXL SHC1 (BIND;CCSB; GRID; HPRD) AXL SHC3 (HPRD) AXL SOCS1 (BIND; HPRD) AXL SRC (CCSB;GRID; HPRD) AXL TENC1 (BIND; GRID; HPRD) BLK BCAS2 (GRID; HPRD) BLK BCL2(GRID; HPRD) BLK BCR (GRID; HPRD) BLK CBL (GRID; HPRD; MINT) BLK CD79A(GRID; HPRD) BLK CD79B (GRID; HPRD) BLK CTR9 (GRID; HPRD) BLK EGFR(MINT) BLK ERBB2 (MINT) BLK FCGR2A (GRID; HPRD) BLK FCGR2B (HPRD) BLKMAX (BIND) BLK MYC (BIND) BLK PLCG2 (GRID; HPRD) BLK TAF1 (BIND) BLKUBE3A (GRID; HPRD) BMX BTK (HPRD) BMX CASP3 (CCSB; GRID; HPRD) BMX CAV1(CCSB; GRID: HPRD) BMX EGFR (MINT) BMX ERBB2 (MINT) BMX ITK (HPRD) BMXKDR (HPRD) BMX PAK1 (GRID; HPRD) BMX PIM1 (IntAct) BMX PTK2 (CCSB; GRID;HPRD) BMX PTPN21 (GRID; HPRD) BMX RAP1A (HPRD) BMX RUFY1 (BIND; CCSB;GRID; HPRD) BMX RUFY2 (HPRD) BMX SRC (CCSB; GRID; HPRD) BMX STAT1 (CCSB;GRID; HPRD) BMX STAT3 (CCSB; GRID; HPRD) BMX STAT5A (CCSB; GRID; HPRD)BMX TEC (HPRD) BMX TNFRSF1B (HPRD) BMX TP53 (IntAct) BTK ARID3A (GRID;HPRD) BTK BLNK (GRID; HPRD; MINT) BTK CAV1 (GRID; HPRD) BTK CBL (BIND;GRID; HPRD) BTK CD19 (HPRD) BTK CMTM3 (HPRD) BTK DAPP1 (HPRD) BTK EWSR1(GRID; HPRD) BTK FAS (GRID; HPRD) BTK FCER1G (IntAct) BTK GNA12 (GRID;HPRD) BTK GNAQ (GRID; HPRD) BTK GNG2 (HPRD) BTK GTF2I (GRID; HPRD;IntAct) BTK HCK (GRID; HPRD) BTK IBTK (GRID; HPRD) BTK IRAK1 (HPRD) BTKITK (HPRD) BTK JAK1 (GRID; HPRD) BTK KHDRBS1 (GRID; HPRD) BTK KIT (HPRD)BTK LYN (GRID; HPRD) BTK MAPK1 (HPRD) BTK MYD88 (HPRD) BTK PIK3AP1(HPRD) BTK PIP4K2A (HPRD) BTK PIP4K2B (HPRD) BTK PIP4K2C (HPRD) BTKPIP5K1A (HPRD) BTK PIP5K1B (HPRD) BTK PIP5K1C (HPRD) BTK PLCG1 (HPRD)BTK PLCG2 (HPRD) BTK PRKCA (HPRD) BTK PRKCB1 (HPRD) BTK PRKCE (HPRD) BTKPRKCQ (HPRD; IntAct) BTK PRKCZ (HPRD) BTK PRKD1 (BIND; GRID; HPRD) BTKRELA (HPRD) BTK SH2B2 (HPRD) BTK SH3BP5 (GRID; HPRD; IntAct) BTK STAT5A(HPRD) BTK SYK (HPRD) BTK TAF1 (BIND) BTK TEC (HPRD) BTK TIRAP (HPRD)BTK TLR4 (HPRD) BTK TLR6 (HPRD) BTK TLR8 (HPRD) BTK TLR9 (HPRD) BTKTNFRSF10A (HPRD) BTK VAV1 (GRID; HPRD) BTK WAS (GRID; HPRD) BTK WASF2(GRID; HPRD) CSF1R CBL (GRID; HPRD) CSF1R CSF1 (GRID; HPRD) CSF1R FYN(GRID; HPRD) CSF1R GRAP2 (GRID; HPRD) CSF1R GRB2 (GRID; HPRD) CSF1RINPP5D (BIND; HPRD) CSF1R INPPL1 (BIND; HPRD) CSF1R LYN (GRID; HPRD)CSF1R PIK3R1 (BIND; GRID; HPRD) CSF1R PIK3R2 (GRID; HPRD) CSF1R RASA1(GRID; HPRD) CSF1R RUNX1 (BIND) CSF1R SHC1 (HPRD) CSF1R SOCS1 (BIND;GRID; HPRD) CSF1R SOCS3 (GRID; HPRD) CSF1R SOS1 (HPRD) CSF1R THOC5(HPRD) CSF1R YES1 (GRID; HPRD) CSK ADRB2 (HPRD) CSK ARRB1 (GRID; HPRD)CSK CAV1 (GRID; HPRD) CSK CBL (GRID) CSK CD247 (GRID; HPRD) CSK CD44(GRID; HPRD) CSK CDH5 (BIND; HPRD) CSK CREBBP (GRID; HPRD) CSK DAB2(GRID; HPRD) CSK DOK1 (HPRD) CSK DOK3 (HPRD) CSK EGFR (BIND; GRID; MINT)CSK ERBB3 (BIND; GRID; MINT) CSK FGR (HPRD) CSK FYN (GRID; HPRD) CSKGJA1 (HPRD; MINT) CSK GSN (HPRD) CSK HCK (HPRD) CSK HNF4A (BIND) CSKHNRPK (GRID; HPRD) CSK IGF1R (BIND; GRID; HPRD; MINT) CSK INSR (BIND;GRID; HPRD) CSK LAIR1 (HPRD) CSK LCK (HPRD) CSK LYN (HPRD) CSK MAPK15(HPRD) CSK PAG1 (GRID; HPRD) CSK PARD3 (MINT) CSK PECAM1 (GRID; HPRD)CSK PLD2 (GRID; HPRD) CSK PRKACA (HPRD) CSK PTK2 (GRID; HPRD) CSK PTPN12(GRID; HPRD) CSK PTPN18 (GRID; HPRD) CSK PTPN22 (HPRD) CSK PXN (GRID;HPRD) CSK RASA1 (GRID; HPRD) CSK RB1 (HPRD) CSK RGS16 (BIND; HPRD) CSKSDC3 (GRID; HPRD) CSK SHC1 (GRID; HPRD) CSK SIT1 (GRID; HPRD) CSK SRC(GRID; HPRD) CSK YES1 (HPRD) CSK YTHDC1 (GRID) DDR1 C1orf165 (HPRD;IntAct) DDR1 COL11A1 (GRID; HPRD) DDR1 COL2A1 (GRID; HPRD) DDR1 COL3A1(GRID; HPRD) DDR1 COL5A2 (GRID; HPRD) DDR1 FRS2 (GRID; HPRD) DDR1 NCK2(HPRD; MINT) DDR1 PLCG1 (GRID; HPRD) DDR1 PTPN11 (HPRD; MINT) DDR1 RGS2(HPRD; IntAct; MDC; MINT) DDR1 SHC1 (GRID; HPRD) DDR1 SNAPIN (HPRD;IntAct; MDC; MINT) DDR1 TM4SF1 (HPRD; IntAct; MDC; MINT) DDR1 TTR (HPRD;IntAct; MDC; MINT) DDR1 WDR57 (HPRD; IntAct; MDC; MINT) DDR2 COL1A1(GRID; HPRD) DDR2 COL3A1 (GRID; HPRD) DDR2 SHC1 (GRID; HPRD; MINT) DDR2SRC (GRID; HPRD; MINT) EGFR ACTA1 (HPRD) EGFR ADAM10 (reactome) EGFRADAM12 (reactome) EGFR ADAM17 (reactome) EGFR ADRBK1 (MINT) EGFR AGTR1(HPRD) EGFR ALCAM (HPRD) EGFR AMH (GRID; HPRD) EGFR ANKS1A (MINT) EGFRANXA1 (HPRD; MINT) EGFR APBA3 (MINT) EGFR APBB1 (MINT) EGFR APBB2 (MINT)EGFR APBB3 (MINT) EGFR APPL1 (MINT) EGFR AR (GRID; HPRD) EGFR AREG (DIP;GRID; HPRD) EGFR ARF4 (GRID; HPRD) EGFR ATP1A1 (HPRD) EGFR ATP1B1 (HPRD)EGFR ATP5C1 (HPRD; IntAct; MDC; MINT) EGFR BTC (DIP; GRID; HPRD) EGFRCALM1 (BIND; HPRD) EGFR CALM2 (HPRD) EGFR CALM3 (HPRD) EGFR CAMK2A(HPRD) EGFR CAMK2G (HPRD) EGFR CAMLG (GRID; HPRD) EGFR CASP1 (GRID;HPRD) EGFR CAV1 (GRID; HPRD) EGFR CAV2 (HPRD) EGFR CAV3 (GRID; HPRD)EGFR CBL (BIND; HPRD; IntAct; MINT) EGFR CBLB (GRID; HPRD) EGFR CBLC(GRID; HPRD) EGFR CD44 (BIND; GRID; HPRD) EGFR CD59 (IntAct) EGFR CD82(GRID; HPRD) EGFR CDC25A (GRID; HPRD) EGFR CDH1 (BIND; GRID; HPRD) EGFRCEACAM1 (HPRD) EGFR CEBPB (GRID; HPRD) EGFR CISH (MINT) EGFR CLTA (BIND;HPRD) EGFR CLTCL1 (HPRD) EGFR COX2 (HPRD) EGFR CRK (BIND; GRID; HPRD;MINT) EGFR CRKL (MINT) EGFR CTNNB1 (GRID; HPRD) EGFR CTNND1 (GRID; HPRD)EGFR DCN (GRID; HPRD) EGFR DEGS1 (GRID; HPRD) EGFR DOK2 (GRID; HPRD)EGFR DOK4 (MINT) EGFR DOK5 (MINT) EGFR DOK6 (MINT) EGFR EGF (BIND; DIP;GRID; HPRD; IntAct; MINT; reactome) EGFR ELF3 (HPRD) EGFR EPB41 (HPRD)EGFR EPHA2 (HPRD) EGFR EPPK1 (GRID; HPRD) EGFR EPS15 (GRID; HPRD) EGFREPS8 (GRID; HPRD) EGFR ERBB2 (BIND; GRID; HPRD; IntAct; MINT) EGFR ERBB3(GRID; HPRD; IntAct) EGFR ERBB4 (HPRD; IntAct) EGFR EREG (GRID; HPRD)EGFR ERRFI1 (GRID; HPRD) EGFR ESR1 (GRID; HPRD) EGFR EZR (HPRD) EGFR FAS(HPRD) EGFR FER (GRID; HPRD) EGFR FES (MINT) EGFR FRS2 (HPRD) EGFR GAB1(GRID; HPRD) EGFR GAB2 (IntAct) EGFR GAPDH (HPRD) EGFR GNAI2 (GRID;HPRD) EGFR GRB10 (BIND; GRID; HPRD; MINT) EGFR GRB14 (GRID; HPRD) EGFRGRB2 (BIND; DIP; GRID; HPRD) EGFR GRB7 (GRID; HPRD) EGFR HBEGF (GRID;HPRD) EGFR HGS (HPRD) EGFR HIST3H3 (HPRD) EGFR HOXC10 (HPRD) EGFRHSP90AA1 (BIND) EGFR HTT (GRID; HPRD) EGFR ICAM1 (HPRD) EGFR INPPL1(GRID; HPRD) EGFR IRS1 (MINT) EGFR IRS4 (MINT) EGFR ITGA5 (GRID; HPRD)EGFR JAK2 (HPRD; MINT) EGFR JUP (GRID) EGFR KRT17 (GRID; HPRD) EGFRKRT18 (GRID; HPRD) EGFR KRT7 (GRID; HPRD) EGFR KRT8 (GRID; HPRD) EGFRLRSAM1 (GRID) EGFR LYN (HPRD) EGFR MAP2K1 (HPRD) EGFR MAP3K14 (GRID;HPRD) EGFR MAP4K1 (GRID; HPRD) EGFR MAPK1 (HPRD) EGFR MAPK8IP1 (MINT)EGFR MAPK8IP2 (MINT) EGFR MET (HPRD) EGFR MIST (MINT) EGFR MUC1 (BIND;GRID; HPRD; MINT) EGFR NCK1 (GRID; HPRD; MINT) EGFR NCK2 (GRID; HPRD)EGFR NRG1 (GRID) EGFR NUMB (MINT) EGFR NUMBL (MINT) EGFR PAK1 (BIND;HPRD) EGFR PDGFRB (GRID; HPRD) EGFR PIK3C2A (IntAct) EGFR PIK3C2B (GRID;HPRD; IntAct) EGFR PIK3R1 (BIND; HPRD; MINT) EGFR PIK3R2 (BIND; HPRD;MINT) EGFR PITPNA (GRID; HPRD) EGFR PKIA (HPRD) EGFR PLCG1 (BIND; GRID;HPRD; MINT) EGFR PLCG2 (MINT) EGFR PLD1 (HPRD) EGFR PLD2 (BIND; GRID;HPRD) EGFR PLEC1 (GRID; HPRD) EGFR PLSCR1 (GRID; HPRD; MINT) EGFR PRKACA(GRID; HPRD) EGFR PRKAR1A (GRID; HPRD) EGFR PRKCA (GRID; HPRD) EGFRPRKCD (HPRD) EGFR PRKD1 (HPRD) EGFR PTK2 (BIND; GRID; HPRD) EGFR PTK2B(GRID; HPRD) EGFR PTK6 (GRID; HPRD; MINT) EGFR PTPN1 (GRID; HPRD) EGFRPTPN11 (BIND; GRID; HPRD; MINT) EGFR PTPN2 (HPRD) EGFR PTPN6 (GRID;HPRD) EGFR PTPRJ (GRID; HPRD) EGFR PTPRS (HPRD) EGFR RASA1 (BIND; GRID;HPRD; MINT) EGFR RGS16 (GRID; HPRD) EGFR RIN2 (MINT) EGFR RIPK1 (GRID;HPRD) EGFR S100A7 (HPRD) EGFR S100A9 (HPRD) EGFR SCAMP1 (BIND; HPRD)EGFR SCAMP3 (BIND; HPRD) EGFR SEC13 (HPRD) EGFR SFN (HPRD) EGFR SGSM2(HPRD; MDC) EGFR SH2B3 (MINT) EGFR SH2D1A (MINT) EGFR SH2D2A (MINT) EGFRSH2D3A (GRID; HPRD) EGFR SH3BGRL (GRID; MINT) EGFR SH3BGRL3 (BIND) EGFRSH3GL2 (HPRD) EGFR SH3KBP1 (HPRD) EGFR SHC1 (BIND; DIP; GRID; HPRD;IntAct; MINT) EGFR SHC2 (MINT) EGFR SHC3 (GRID; HPRD; MINT) EGFR SLC3A2(HPRD) EGFR SNRPD2 (GRID; HPRD) EGFR SNX1 (DIP; GRID; HPRD) EGFR SNX2(GRID; HPRD) EGFR SNX4 (GRID; HPRD) EGFR SNX6 (GRID; HPRD) EGFR SOCS1(GRID; HPRD) EGFR SOCS3 (GRID; HPRD) EGFR SOS1 (GRID; HPRD) EGFR SOS2(GRID; HPRD) EGFR SRC (GRID; HPRD; reactome) EGFR STAM2 (HPRD) EGFRSTAT1 (GRID; HPRD) EGFR STAT3 (BIND; GRID; HPRD) EGFR STAT5A (GRID;HPRD) EGFR STAT5B (BIND; GRID; HPRD; MINT) EGFR SYK (MINT) EGFR TGFA(DIP; GRID; HPRD; IntAct) EGFR TJP1 (GRID; HPRD) EGFR TLN1 (MINT) EGFRTLR2 (MINT) EGFR TLR4 (MINT) EGFR TNC (GRID; HPRD) EGFR TNK2 (GRID;HPRD) EGFR TNS4 (MINT) EGFR TUBA4A (HPRD) EGFR UBB (HPRD) EGFR VAV1(GRID; HPRD) EGFR VAV2 (BIND; GRID; HPRD) EGFR VAV3 (GRID; HPRD) EGFRXPO6 (HPRD) EGFR XRCC6 (GRID; HPRD) EGFR YWHAZ (BIND; HPRD) EGFR ZAP70(MINT) EGFR ZNF259 (GRID; HPRD) EPHA1 EFNA1 (BIND; GRID; HPRD) EPHA1HNF4A (BIND) EPHA1 ONECUT1 (BIND) EPHA1 SMURF2 (BIND; HPRD) EPHA2 ACP1(GRID; HPRD) EPHA2 CBL (HPRD) EPHA2 CDH5 (HPRD) EPHA2 CLDN4 (HPRD) EPHA2EFNA1 (BIND; DIP; GRID; HPRD) EPHA2 EFNA2 (GRID; HPRD) EPHA2 EFNA3(BIND; GRID; HPRD) EPHA2 EFNA4 (BIND; GRID; HPRD) EPHA2 EFNA5 (BIND;GRID; HPRD) EPHA2 GRB2 (GRID; HPRD) EPHA2 HNF4A (BIND) EPHA2 PAX3 (BIND)EPHA2 PIK3R1 (GRID) EPHA2 PIK3R2 (HPRD) EPHA2 PTK2 (GRID; HPRD) EPHA2PTPN11 (GRID; HPRD) EPHA2 RASA1 (HPRD) EPHA2 SHC1 (GRID; HPRD) EPHA2 SLA(GRID; HPRD) EPHA2 TIAM1 (HPRD) EPHA2 TNFAIP1 (GRID; HPRD) EPHA3 ADAM10(HPRD) EPHA3 CRK (GRID; HPRD) EPHA3 E2F4 (BIND) EPHA3 EFNA1 (HPRD) EPHA3EFNA2 (GRID; HPRD) EPHA3 EFNA5 (GRID; HPRD) EPHA3 EFNB2 (GRID; HPRD)EPHA3 RUFY1 (GRID; HPRD) EPHA3 RUFY2 (GRID; HPRD) EPHA3 TP53 (GRID)EPHA4 ARHGEF15 (CCSB; GRID; HPRD) EPHA4 EFNA1 (BIND; CCSB; GRID; HPRD)EPHA4 EFNA3 (BIND; GRID; HPRD) EPHA4 EFNA4 (BIND; GRID; HPRD) EPHA4EFNA5 (BIND; HPRD) EPHA4 EFNB2 (BIND; GRID; HPRD) EPHA4 EFNB3 (GRID;HPRD) EPHA4 FGFR1 (HPRD) EPHA4 FGFR2 (HPRD) EPHA4 FGFR3 (HPRD) EPHA4FGFR4 (HPRD) EPHA4 FYN (CCSB; GRID; HPRD) EPHA4 NGEF (CCSB; GRID; HPRD)EPHA4 PAX3 (BIND) EPHA5 EFNA1 (BIND; GRID; HPRD) EPHA5 EFNA2 (GRID;HPRD) EPHA5 EFNA3 (BIND; GRID; HPRD) EPHA5 EFNA4 (BIND; HPRD) EPHA5EFNA5 (BIND; GRID; HPRD) EPHA5 STAT3 (GRID) EPHA6 EFNA1 (BIND; GRID;HPRD) EPHA6 EFNA3 (BIND) EPHA6 EFNA4 (BIND; HPRD) EPHA6 EFNA5 (BIND)EPHA7 EFNA1 (BIND: CCSB; GRID; HPRD) EPHA7 EFNA3 (BIND; HPRD) EPHA7EFNA4 (BIND; HPRD) EPHA7 EFNA5 (BIND; HPRD) EPHA7 MLLT4 (BIND; GRID;HPRD; MINT) EPHA7 SDCBP (BIND) EPHA8 EFNA1 (HPRD) EPHA8 EFNA4 (HPRD)EPHA8 EFNA5 (GRID; HPRD) EPHA8 FYN (CCSB; GRID; HPRD) EPHA8 PIK3CG(CCSB; GRID; HPRD) EPHB1 ACP1 (BIND; GRID; HPRD) EPHB1 EFNA1 (HPRD)EPHB1 EFNA5 (GRID; HPRD) EPHB1 EFNB1 (BIND; GRID; HPRD) EPHB1 EFNB2(BIND; GRID; HPRD) EPHB1 EPHB6 (GRID; HPRD) EPHB1 GRB10 (BIND; HPRD;IntAct) EPHB1 GRB2 (BIND; GRID; HPRD) EPHB1 NCK1 (GRID; HPRD) EPHB1PDGFRB (BIND) EPHB1 PXN (HPRD) EPHB2 ACP1 (BIND; GRID; HPRD) EPHB2ADAM17 (GRID) EPHB2 AQP1 (HPRD) EPHB2 ARHGEF6 (HPRD) EPHB2 BCL2 (GRID)EPHB2 EFNA5 (HPRD) EPHB2 EFNB1 (BIND; GRID; HPRD) EPHB2 EFNB2 (BIND;GRID; HPRD) EPHB2 ERF (HPRD) EPHB2 FOS (HPRD) EPHB2 GRB2 (HPRD) EPHB2GRIN1 (HPRD) EPHB2 GRIN2B (HPRD) EPHB2 ITSN1 (GRID; HPRD) EPHB2 KALRN(HPRD) EPHB2 MLLT4 (BIND; GRID; HPRD; MINT) EPHB2 NCK1 (GRID; HPRD)EPHB2 PICK1 (HPRD) EPHB2 PTK2 (HPRD) EPHB2 RASA1 (GRID; HPRD) EPHB2 RRAS(HPRD) EPHB2 RYK (GRID; HPRD) EPHB2 SDC2 (HPRD) EPHB2 SDCBP (HPRD) EPHB2SH2D3C (HPRD) EPHB2 SRC (GRID; HPRD) EPHB2 SYNJ1 (HPRD) EPHB2 VAV2(HPRD) EPHB3 CRK (GRID; HPRD) EPHB3 EFNB3 (GRID; HPRD) EPHB3 FYN (GRID;HPRD) EPHB3 HNRPA3 (BIND) EPHB3 MLLT4 (BIND; GRID; HPRD; MINT) EPHB3RASA1 (GRID; HPRD) EPHB3 RYK (GRID; HPRD) EPHB4 EFNB2 (GRID; HPRD) EPHB4GRIN1 (HPRD) EPHB4 SDC3 (HPRD) EPHB6 CBL (GRID; HPRD) EPHB6 CRK (HPRD)EPHB6 CRKL (HPRD) EPHB6 EFNB2 (GRID; HPRD) EPHB6 GRB2 (HPRD) EPHB6 HDHD2(HPRD) EPHB6 HNRPA3 (BIND) EPHB6 MLLT4 (BIND; GRID; HPRD; MINT) EPHB6SAT1 (HPRD; IntAct; MDC; MINT) ERBB2 ACPP (HPRD) ERBB2 ANKS1A (MINT)ERBB2 APBB1 (MINT) ERBB2 APBB3 (MINT) ERBB2 BLNK (MINT) ERBB2 BTC (GRID;HPRD) ERBB2 CAV1 (GRID; HPRD) ERBB2 CBL (BIND) ERBB2 CD82 (GRID; HPRD)ERBB2 CDC37 (BIND) ERBB2 CHN1 (MINT) ERBB2 CISH (MINT) ERBB2 CRK (MINT)ERBB2 CRKL (MINT) ERBB2 CTNNB1 (GRID; HPRD) ERBB2 DAB1 (MINT) ERBB2 DLG4(GRID; HPRD) ERBB2 DOK1 (MINT) ERBB2 DOK4 (MINT) ERBB2 DOK6 (MINT) ERBB2EGF (GRID; HPRD) ERBB2 ERBB2IP (GRID; HPRD) ERBB2 ERBB3 (GRID; HPRD;IntAct) ERBB2 ERBB4 (IntAct) ERBB2 ERRFI1 (BIND; GRID; HPRD) ERBB2 ESR1(HPRD) ERBB2 FER (MINT) ERBB2 FGR (MINT) ERBB2 GRAP2 (MINT) ERBB2 GRB2(BIND; GRID; HPRD) ERBB2 GRB7 (GRID; HPRD; MINT) ERBB2 H3F3A (BIND)ERBB2 HLA-A (GRID; HPRD) ERBB2 HSP90AA1 (BIND; GRID; HPRD) ERBB2 HSP90B1(GRID; HPRD) ERBB2 HSPA8 (BIND) ERBB2 IL6ST (GRID; HPRD) ERBB2 IRS1(MINT) ERBB2 IRS4 (MINT) ERBB2 ITGA5 (HPRD) ERBB2 ITGB4 (GRID; HPRD)ERBB2 ITK (MINT) ERBB2 JAK1 (MINT) ERBB2 JAK2 (GRID; HPRD) ERBB2 JUP(GRID; HPRD) ERBB2 MAPK8IP1 (MINT) ERBB2 MAPK8IP2 (MINT) ERBB2 MATK(GRID; HPRD; MINT) ERBB2 MIST (MINT) ERBB2 MMP16 (BIND) ERBB2 MUC1(GRID; HPRD) ERBB2 MUC4 (HPRD) ERBB2 NCK2 (MINT) ERBB2 NCOA3 (BIND)ERBB2 NCOR1 (BIND) ERBB2 NF2 (HPRD) ERBB2 NRG1 (DIP; GRID; MINT) ERBB2PAK1 (GRID; HPRD) ERBB2 PICK1 (GRID; HPRD) ERBB2 PIK3C2A (IntAct) ERBB2PIK3C2B (IntAct) ERBB2 PIK3R1 (GRID; HPRD; MINT) ERBB2 PIK3R2 (GRID;HPRD; MINT) ERBB2 PLCG1 (GRID; HPRD; MINT) ERBB2 PLCG2 (MINT) ERBB2 PTK2(GRID; HPRD) ERBB2 PTK2B (GRID; HPRD) ERBB2 PTPN11 (BIND; GRID; MINT)ERBB2 PTPN18 (HPRD) ERBB2 RASA1 (MINT) ERBB2 RIN1 (MINT) ERBB2 RIN2(MINT) ERBB2 SERPINA3 (GRID; HPRD) ERBB2 SH2B2 (MINT) ERBB2 SH2B3 (MINT)ERBB2 SH2D1B (MINT) ERBB2 SH2D2A (MINT) ERBB2 SH3BGRL (GRID) ERBB2SH3BGRL3 (BIND; MINT) ERBB2 SH3BP2 (MINT) ERBB2 SHC1 (BIND; GRID; HPRD;MINT) ERBB2 SHC2 (MINT) ERBB2 SHC3 (MINT) ERBB2 SLA2 (MINT) ERBB2 SOCS1(MINT) ERBB2 SOS1 (GRID; HPRD) ERBB2 SRC (BIND; GRID; HPRD) ERBB2 STAT1(MINT) ERBB2 STAT3 (MINT) ERBB2 STUB1 (GRID; HPRD) ERBB2 SUPT6H (MINT)ERBB2 SYK (MINT) ERBB2 TAF1 (BIND) ERBB2 TEC (MINT) ERBB2 TGFA (HPRD)ERBB2 TLN1 (MINT) ERBB2 TOB1 (GRID; HPRD) ERBB2 TP53RK (BIND) ERBB2 TXK(MINT) ERBB2 UBB (HPRD) ERBB2 VAV2 (MINT) ERBB2 VAV3 (MINT) ERBB3 AGTR2(HPRD) ERBB3 CD82 (CCSB; GRID; HPRD) ERBB3 CDK5 (HPRD) ERBB3 CHN2 (MINT)ERBB3 CRK (MINT) ERBB3 CRKL (MINT) ERBB3 DAB1 (MINT) ERBB3 DAPP1 (MINT)ERBB3 EGF (GRID; HPRD) ERBB3 EGR1 (GRID) ERBB3 ERBB4 (HPRD; IntAct)ERBB3 FER (MINT) ERBB3 FGFR1 (MINT) ERBB3 FLYWCH1 (HPRD; MDC) ERBB3 GRB2(BIND; GRID; HPRD) ERBB3 GRB7 (CCSB; GRID; HPRD; MINT) ERBB3 HCK (MINT)ERBB3 HNF4A (BIND) ERBB3 IL6ST (GRID; HPRD) ERBB3 ITK (MINT) ERBB3 JAK2(MINT) ERBB3 JAK3 (MINT) ERBB3 MUC1 (GRID: HPRD) ERBB3 NCK1 (MINT) ERBB3NCK2 (MINT) ERBB3 NRG1 (BIND; CCSB; DIP; GRID; HPRD; MINT) ERBB3 NRG2(GRID; HPRD) ERBB3 ODF2L (HPRD; MDC) ERBB3 PA2G4 (CCSB; GRID; HPRD;IntAct) ERBB3 PIK3R1 (GRID; HPRD; IntAct; MINT) ERBB3 PIK3R2 (BIND;GRID; MINT) ERBB3 PLCG1 (MINT) ERBB3 PTGES3 (CCSB; GRID; HPRD) ERBB3PTK2 (CCSB; GRID; HPRD) ERBB3 PTK2B (HPRD) ERBB3 PTK6 (GRID; HPRD; MINT)ERBB3 RASA1 (MINT) ERBB3 RASA4 (HPRD; IntAct; MDC; MINT) ERBB3 RGS4(CCSB; GRID; HPRD) ERBB3 RIN1 (MINT) ERBB3 RNF41 (HPRD) ERBB3 RPN1(GRID) ERBB3 SH2B3 (MINT) ERBB3 SH2D1A (MINT) ERBB3 SHC1 (BIND; CCSB;GRID; HPRD; MINT) ERBB3 SHC3 (MINT) ERBB3 SOS1 (GRID; HPRD; MINT) ERBB3SRC (MINT) ERBB3 SYK (MINT) ERBB3 TNS4 (MINT) ERBB3 TXK (MINT) ERBB3VAV2 (MINT) ERBB3 VAV3 (MINT) ERBB3 ZAP70 (MINT) ERBB3 ZNF207 (GRID)ERBB4 ADAM17 (HPRD) ERBB4 ANKS1A (MINT) ERBB4 BTC (GRID; HPRD) ERBB4CD44 (GRID; HPRD) ERBB4 CRK (BIND; GRID) ERBB4 CRKL (MINT) ERBB4 CTGF(GRID; HPRD) ERBB4 DLG1 (GRID; HPRD) ERBB4 DLG2 (BIND; GRID; HPRD;IntAct) ERBB4 DLG3 (BIND; GRID; HPRD; IntAct) ERBB4 DLG4 (BIND; GRID;HPRD; IntAct; MINT) ERBB4 EREG (GRID; HPRD) ERBB4 GRB2 (BIND; GRID)ERBB4 GRIN1 (IntAct) ERBB4 HBEGF (GRID; HPRD) ERBB4 MUC1 (GRID; HPRD)ERBB4 NCK1 (GRID) ERBB4 NCK2 (BIND; MINT) ERBB4 NRG1 (DIP; GRID; HPRD;MINT) ERBB4 NRG2 (GRID; HPRD) ERBB4 NRG3 (HPRD) ERBB4 NRG4 (HPRD) ERBB4PIK3R2 (BIND; GRID; MINT) ERBB4 PTPN11 (BIND; GRID; MINT) ERBB4 RASA1(MINT) ERBB4 RNF41 (GRID; HPRD) ERBB4 SHC1 (BIND; GRID; MINT) ERBB4SNTB2 (BIND; GRID; HPRD; IntAct) ERBB4 STAT5A (GRID; HPRD) ERBB4 STAT5B(BIND; MINT) ERBB4 SYK (MINT) ERBB4 TGFA (HPRD) ERBB4 YAP1 (GRID; HPRD)FER CALM1 (HPRD) FER CTNNB1 (HPRD) FER CTNND1 (GRID; HPRD) FER CTTN(GRID; HPRD) FER IRS1 (HPRD) FER JAK1 (HPRD) FER JUP (HPRD) FER STAT3(GRID; HPRD) FER TMF1 (GRID; HPRD) FER YWHAB (HPRD) FES BCAR1 (GRID;HPRD) FES BCR (GRID; HPRD) FES CSF2RB (HPRD) FES DOK1 (HPRD) FES DPYSL5(CCSB; GRID; HPRD) FES HSH2D (GRID; HPRD) FES IL4R (GRID; HPRD) FES IRS1(HPRD) FES IRS2 (HPRD) FES JAK1 (HPRD) FES JAK2 (HPRD) FES JAK3 (HPRD)FES PIK3R1 (GRID; HPRD) FES PLXNA1 (HPRD) FES PSMD13 (GRID) FES RASA1(CCSB; GRID; HPRD) FES RASA3 (GRID; HPRD) FES STAT3 (CCSB; GRID; HPRD)FGFR1 ATF2 (BIND) FGFR1 BNIP2 (BIND; CCSB; GRID; HPRD) FGFR1 CBL(reactome) FGFR1 CREBBP (HPRD) FGFR1 CRK (HPRD) FGFR1 FDPS (CCSB; GRID;HPRD) FGFR1 FGF1 (CCSB; DIP; GRID; HPRD; MINT; reactome) FGFR1 FGF17(HPRD; reactome) FGFR1 FGF18 (HPRD) FGFR1 FGF2 (DIP; GRID; HPRD; IntAct;reactome) FGFR1 FGF20 (reactome) FGFR1 FGF23 (reactome) FGFR1 FGF3(HPRD) FGFR1 FGF4 (HPRD; reactome) FGFR1 FGF5 (DIP; GRID; HPRD;reactome) FGFR1 FGF6 (HPRD; reactome) FGFR1 FGF7 (HPRD) FGFR1 FGF8(HPRD; reactome) FGFR1 FGF9 (HPRD; reactome) FGFR1 FGFR2 (BIND) FGFR1FRS2 (CCSB; GRID; HPRD; reactome) FGFR1 FRS3 (BIND; GRID; HPRD;reactome) FGFR1 GRB14 (GRID; HPRD) FGFR1 GRB2 (CCSB; GRID; HPRD;reactome) FGFR1 IL17RD (HPRD) FGFR1 JUN (BIND) FGFR1 KL (reactome) FGFR1KPNB1 (GRID; HPRD) FGFR1 MMP2 (HPRD) FGFR1 NCAM1 (GRID; HPRD) FGFR1 NCK2(BIND; HPRD) FGFR1 NRP1 (HPRD) FGFR1 PIK3R1 (HPRD) FGFR1 PIK3R2 (BIND;HPRD) FGFR1 PLCG1 (BIND; HPRD) FGFR1 RPS6KA1 (BIND; HPRD) FGFR1 RTN1(BIND) FGFR1 RTN3 (BIND; HPRD) FGFR1 SH3BP2 (BIND; HPRD) FGFR1 SHB(BIND; GRID) FGFR1 SHC1 (CCSB; GRID; HPRD) FGFR1 SLA (BIND; HPRD) FGFR1SOS1 (HPRD) FGFR1 TENC1 (BIND; HPRD) FGFR2 CACNA1D (GRID; HPRD) FGFR2CBL (reactome) FGFR2 FGF1 (DIP; GRID; HPRD; IntAct; MINT; reactome)FGFR2 FGF10 (DIP; GRID; HPRD; IntAct; MINT; reactome) FGFR2 FGF16(reactome) FGFR2 FGF17 (HPRD; reactome) FGFR2 FGF18 (HPRD; reactome)FGFR2 FGF2 (DIP; GRID; IntAct; reactome) FGFR2 FGF20 (reactome) FGFR2FGF22 (reactome) FGFR2 FGF23 (HPRD; reactome) FGFR2 FGF3 (DIP; HPRD;reactome) FGFR2 FGF4 (HPRD; reactome) FGFR2 FGF5 (GRID; HPRD; reactome)FGFR2 FGF6 (DIP; HPRD; reactome) FGFR2 FGF7 (DIP; GRID; HPRD; reactome)FGFR2 FGF8 (HPRD; reactome) FGFR2 FGF9 (DIP; GRID; HPRD; reactome) FGFR2FRS2 (reactome) FGFR2 FRS3 (reactome) FGFR2 FYN (HPRD) FGFR2 GRB2(reactome) FGFR2 ITGA5 (HPRD) FGFR2 PAK4 (HPRD) FGFR2 PLCG1 (GRID; HPRD)FGFR2 UQCRB (BIND) FGFR3 ATF3 (HPRD; IntAct; MDC) FGFR3 C13orf34 (HPRD;IntAct; MDC) FGFR3 C6orf47 (GRID; HPRD) FGFR3 CBL (reactome) FGFR3CCDC17 (HPRD; IntAct; MDC) FGFR3 CENTD2 (HPRD; IntAct; MDC) FGFR3 CHGB(HPRD; IntAct; MDC) FGFR3 CTSK (HPRD; IntAct; MDC) FGFR3 FGF1 (DIP;GRID; HPRD; IntAct; reactome) FGFR3 FGF16 (reactome) FGFR3 FGF17 (HPRD;reactome) FGFR3 FGF18 (HPRD; reactome) FGFR3 FGF2 (DIP; HPRD; reactome)FGFR3 FGF20 (reactome) FGFR3 FGF23 (HPRD; reactome) FGFR3 FGF3 (HPRD)FGFR3 FGF4 (DIP; HPRD; reactome) FGFR3 FGF5 (DIP; HPRD; reactome) FGFR3FGF6 (HPRD) FGFR3 FGF7 (DIP; HPRD) FGFR3 FGF8 (GRID; HPRD; reactome)FGFR3 FGF9 (GRID; HPRD; reactome) FGFR3 FGFR2 (BIND) FGFR3 FRS2(reactome) FGFR3 FRS3 (reactome) FGFR3 GPSM3 (BIND; HPRD; IntAct; MINT)FGFR3 GRB2 (GRID; HPRD; reactome) FGFR3 GTF3C1 (HPRD; MDC) FGFR3 HBZ(HPRD; IntAct; MDC) FGFR3 HNRNPL (HPRD; IntAct; MDC) FGFR3 KIAA1377(HPRD; IntAct; MDC) FGFR3 KRT8 (HPRD; IntAct; MDC) FGFR3 NDUFS6 (HPRD;IntAct; MDC) FGFR3 POLA2 (HPRD; IntAct; MDC) FGFR3 PTPN11 (GRID) FGFR3RADIL (HPRD; MDC) FGFR3 RNF130 (HPRD; IntAct; MDC) FGFR3 RPL8 (HPRD;IntAct; MDC) FGFR3 SH2B1 (GRID; HPRD) FGFR3 SLC25A6 (HPRD; IntAct; MDC)FGFR3 SMA4 (IntAct) FGFR3 SMG7 (HPRD; MDC) FGFR3 STAT1 (HPRD) FGFR3STAT3 (HPRD) FGFR4 ADH6 (BIND) FGFR4 ATF2 (BIND) FGFR4 ATP5H (BIND)FGFR4 CALM1 (BIND) FGFR4 CBL (reactome) FGFR4 CDH2 (HPRD) FGFR4 COX6C(BIND) FGFR4 COX7B (BIND) FGFR4 DPM2 (BIND) FGFR4 E2F4 (BIND) FGFR4EIF3E (BIND) FGFR4 FBP1 (BIND) FGFR4 FGA (BIND) FGFR4 FGF1 (DIP; GRID;HPRD; reactome) FGFR4 FGF16 (reactome) FGFR4 FGF17 (HPRD; reactome)FGFR4 FGF18 (HPRD; reactome) FGFR4 FGF19 (BIND; DIP; GRID; HPRD;reactome) FGFR4 FGF2 (DIP; GRID; HPRD; reactome) FGFR4 FGF20 (reactome)FGFR4 FGF23 (reactome) FGFR4 FGF3 (HPRD) FGFR4 FGF4 (HPRD; reactome)FGFR4 FGF5 (HPRD) FGFR4 FGF6 (GRID; HPRD; reactome) FGFR4 FGF7 (HPRD)FGFR4 FGF8 (GRID; HPRD; reactome) FGFR4 FGF9 (HPRD; reactome) FGFR4 FN1(BIND) FGFR4 FRS2 (reactome) FGFR4 FRS3 (reactome) FGFR4 GJC1 (BIND)FGFR4 GLRX (BIND) FGFR4 GPT (BIND) FGFR4 GRB2 (reactome) FGFR4 GSTO1(BIND) FGFR4 HNRNPC (BIND) FGFR4 LDLRAP1 (BIND) FGFR4 PLCG1 (HPRD) FGFR4POLR2I (BIND) FGFR4 PPIA (BIND) FGFR4 PSMA4 (BIND) FGFR4 RPLP2 (BIND)FGFR4 SF3B4 (BIND) FGFR4 STAT1 (HPRD) FGFR4 STAT3 (HPRD) FGFR4 TLR4(BIND) FGR ARRB1 (GRID; HPRD) FGR CBL (GRID; HPRD) FGR CCR3 (GRID; HPRD)FGR CD24 (GRID; HPRD) FGR DAB2 (GRID; HPRD) FGR DOK1 (HPRD) FGR HCLS1(HPRD) FGR HSP90AA1 (DIP) FGR INPP5D (GRID; HPRD) FGR NCOA6 (GRID) FGRPLAUR (DIP) FGR PTK2 (GRID; HPRD) FGR SLAMF1 (GRID; HPRD) FGR SNCA(HPRD) FGR SRC (HPRD) FGR SYK (GRID; HPRD; MINT) FGR VDR (HPRD) FGR WAS(GRID; HPRD; MINT) FGR YWHAQ (GRID; HPRD) FLT1 ATR (GRID; HPRD) FLT1 CRK(HPRD) FLT1 CTNNB1 (HPRD) FLT1 FYN (HPRD) FLT1 GRB2 (HPRD) FLT1 KDR(GRID; HPRD) FLT1 NCK1 (BIND; HPRD) FLT1 NRP1 (HPRD) FLT1 NRP2 (HPRD)FLT1 PGF (DIP; GRID; HPRD; IntAct) FLT1 PIK3R1 (BIND) FLT1 PLCG1 (BIND;HPRD) FLT1 PLCG2 (GRID; HPRD) FLT1 PTK2 (HPRD) FLT1 PTPN11 (BIND; HPRD)FLT1 SHC1 (GRID; HPRD) FLT1 SHC2 (MINT) FLT1 STAT1 (HPRD) FLT1 STAT3(HPRD) FLT1 VEGFA (BIND; DIP; GRID; HPRD; IntAct) FLT1 VEGFB (GRID;HPRD) FLT1 YES1 (HPRD) FLT3 CBLB (GRID; HPRD) FLT3 FIZ1 (HPRD) FLT3FLT3LG (GRID; HPRD) FLT3 GRB2 (GRID; HPRD) FLT3 INPP5D (HPRD) FLT3 NCK1(HPRD) FLT3 PTPN12 (HPRD) FLT3 PTPN6 (HPRD) FLT3 SH3BP2 (HPRD) FLT3 SHC1(GRID; HPRD) FLT3 SOCS1 (GRID; HPRD) FLT4 ATF7IP (BIND; HPRD) FLT4 FIGF(DIP; GRID; HPRD) FLT4 GRB2 (GRID; HPRD) FLT4 ITGA5 (HPRD) FLT4 ITGB1(GRID; HPRD) FLT4 KDR (HPRD; IntAct) FLT4 PTK2 (HPRD) FLT4 PTPN11 (HPRD)FLT4 SHC1 (GRID; HPRD) FLT4 SHC3 (HPRD) FLT4 VEGFC [BIND; DIP; GRID;HPRD) FRK HNF4A (BIND) FRK RB1 (BIND; CCSB; GRID; HPRD) FYN ACP1 (HPRD)FYN ADAM15 (CCSB; GRID; HPRD; MINT) FYN ADD2 (GRID; HPRD) FYN ATXN1(HPRD; IntAct; MINT) FYN BCAR1 (GRID; HPRD) FYN BCL3 (GRID; HPRD) FYNCASP3 (HPRD) FYN CASP8 (HPRD) FYN CAV1 (HPRD) FYN CBL (BIND; GRID; HPRD;MINT) FYN CBLB (HPRD) FYN CD19 (GRID; HPRD) FYN CD2 (BIND; CCSB; GRID;HPRD; MINT) FYN CD226 (GRID; HPRD) FYN CD247 (BIND; CCSB; GRID; HPRD)FYN CD2AP (GRID; HPRD; MINT) FYN CD36 (GRID; HPRD) FYN CD44 (CCSB; GRID;HPRD) FYN CD48 (CCSB; GRID) FYN CD5 (HPRD) FYN CD55 (CCSB; GRID; HPRD)FYN CD79A (HPRD) FYN CD79B (HPRD) FYN CDC2 (CCSB; GRID; HPRD) FYN CDK5(HPRD) FYN CNN1 (HPRD) FYN CNN3 (HPRD) FYN CNTN1 (GRID; HPRD) FYNCNTNAP1 (HPRD) FYN CRK (HPRD) FYN CSF2RB (HPRD) FYN CTLA4 (GRID; HPRD)FYN CTNNB1 (HPRD) FYN CTNND1 (GRID) FYN CTNND2 (HPRD) FYN DCC (BIND) FYNDLG4 (GRID; HPRD) FYN DOK1 (HPRD) FYN DOK3 (HPRD) FYN DOK4 (BIND; HPRD)FYN EVL (CCSB; GRID; HPRD) FYN FAS (CCSB; GRID; HPRD) FYN FASLG (GRID;HPRD) FYN FCER2 (BIND; CCSB; GRID; HPRD) FYN FCGR2A (HPRD) FYN FLOT1(HPRD) FYN FLOT2 (HPRD) FYN FN1 (HPRD) FYN FNBP4 (CCSB; GRID; HPRD) FYNFYB (GRID; HPRD; MINT) FYN GAB3 (GRID; HPRD) FYN GNB2L1 (CCSB; GRID;HPRD) FYN GP6 (GRID; HPRD) FYN GRAP (HPRD) FYN GRB10 (GRID; HPRD) FYNGRIN2A (GRID; HPRD) FYN GRIN2B (GRID; HPRD) FYN HNRPK (BIND; CCSB; GRID;HPRD) FYN HRAS (HPRD; MINT) FYN HTR6 (IntAct) FYN IL2RB (HPRD) FYN IL7R(BIND; GRID; HPRD) FYN IRS1 (HPRD) FYN ITGAV (HPRD) FYN ITGB4 (HPRD) FYNITGB6 (HPRD) FYN ITK (GRID; HPRD) FYN ITPR1 (HPRD) FYN JAK2 (GRID; HPRD)FYN JUP (HPRD) FYN KDR (HPRD; MINT) FYN KHDRBS1 (GRID; HPRD; IntAct;MINT) FYN KIT (HPRD) FYN LCP2 (HPRD) FYN LYN (HPRD) FYN MAG (GRID; HPRD)FYN MAP2 (HPRD; MINT) FYN MAPT (HPRD) FYN MCAM (GRID; HPRD) FYN MS4A1(CCSB; GRID; HPRD) FYN NCAM1 (GRID; HPRD) FYN NCK1 (MINT) FYN NEDD9(GRID; HPRD) FYN NMT1 (HPRD) FYN NPHS1 (GRID; HPRD) FYN NTRK2 (GRID;HPRD) FYN PAG1 (GRID; HPRD) FYN PDE4D (BIND) FYN PDGFRB (CCSB; GRID;HPRD) FYN PECAM1 (GRID; HPRD) FYN PIK3R1 (BIND; GRID; HPRD) FYN PIK3R2(GRID; HPRD) FYN PLAUR (CCSB; GRID; HPRD) FYN PLCG1 (GRID; HPRD) FYNPLCG2 (CCSB; GRID; HPRD) FYN PLD2 (GRID; HPRD) FYN PRKCD (HPRD; IntAct)FYN PRKCE (HPRD) FYN PRKCH (HPRD) FYN PRKCQ (GRID; HPRD) FYN PRKCZ(HPRD) FYN PTK2 (GRID; HPRD) FYN PTPN11 (CCSB; GRID; HPRD) FYN PTPN5(GRID; HPRD) FYN PTPRA [CCSB; GRID; HPRD) FYN PTPRC (GRID; HPRD) FYNPTPRE (HPRD) FYN PTPRF (HPRD) FYN PTPRZ1 (HPRD) FYN PXN (GRID; HPRD) FYNRAF1 (CCSB; GRID; HPRD) FYN RICS (GRID; HPRD) FYN SDC3 (CCSB; GRID;HPRD) FYN SH2B2 (HPRD) FYN SH2D1A (GRID; HPRD; MINT) FYN SH3BP2 (HPRD)FYN SHC1 (HPRD; MINT) FYN SIT1 (GRID; HPRD) FYN SKAP1 (GRID; HPRD) FYNSKAP2 (CCSB; GRID; HPRD) FYN SLAMF1 (CCSB; GRID; HPRD) FYN SNCA (GRID;HPRD) FYN SNX26 (HPRD) FYN SOCS1 (BIND; GRID; HPRD) FYN SOS1 (GRID;HPRD) FYN SPHK1 (IntAct) FYN SPHK2 (IntAct) FYN SPN (GRID; HPRD) FYN SRC(MINT) FYN STAT1 (HPRD) FYN SYK (CCSB; GRID; HPRD) FYN TEK (HPRD) FYNTHY1 (CCSB; GRID; HPRD) FYN TNK2 (CCSB; GRID; HPRD) FYN TOM1L1 (HPRD)FYN TRAT1 (HPRD) FYN TRPC6 (GRID; HPRD) FYN TRPV4 (GRID; HPRD) FYNTUBA1B (CCSB; GRID; HPRD) FYN TUBA3C (CCSB; GRID; HPRD) FYN TUBA4A(CCSB; GRID; HPRD) FYN TXK (HPRD) FYN TYK2 (CCSB; GRID; HPRD) FYN TYRO3(GRID; HPRD) FYN UNC119 (CCSB; GRID; HPRD) FYN VAV1 (CCSB; GRID; HPRD)FYN WAS (CCSB; GRID; HPRD; MINT) FYN WASF1 (GRID; HPRD) FYN WASF2 (GRID;HPRD) FYN YTHDC1 (GRID; HPRD) FYN ZAP70 (CCSB; GRID; HPRD) HCK ACTB(HPRD) HCK ADAM15 (CCSB; GRID; HPRD; MINT) HCK AGK (HPRD; MDC) HCK ARRB1(HPRD) HCK BCAR1 (GRID; HPRD) HCK BCR (GRID; HPRD) HCK C14orf4 (HPRD)HCK C2orf44 (HPRD; IntAct) HCK CBL (GRID; HPRD) HCK CCR3 (GRID; HPRD)HCK CD2AP (HPRD; IntAct) HCK CSF2RB (HPRD) HCK CSF3R (GRID; HPRD) HCKDDEF1 (HPRD; IntAct) HCK DNM2 (HPRD; IntAct) HCK DOK1 (HPRD) HCK ELMO1(GRID; HPRD; IntAct; MINT) HCK EVL (HPRD; IntAct) HCK FCGR1A (CCSB;GRID; HPRD) HCK FCGR2A (CCSB; GRID; HPRD) HCK GALNAC4S-6ST (GRID; HPRD)HCK HNRPK (HPRD) HCK IL6ST (GRID; HPRD) HCK INPP5D (HPRD) HCK KHDRBS1(BIND; HPRD; IntAct) HCK KIT (HPRD) HCK LCP2 (HPRD; IntAct) HCK PECAM1(GRID; HPRD) HCK PIK3CB (HPRD) HCK PIK3R1 (HPRD; IntAct) HCK PIK3R2(HPRD; IntAct) HCK PLAUR (CCSB; DIP; GRID; HPRD) HCK PLCG1 (GRID; HPRD)HCK PLCG2 (HPRD) HCK RAPGEF1 (GRID; HPRD) HCK RASA1 (CCSB; GRID; HPRD)HCK RASA3 (GRID; HPRD) HCK SF3B3 (HPRD; IntAct) HCK SH3BP1 [HPRD;IntAct) HCK SH3KBP1 (HPRD; IntAct) HCK SKAP2 (CCSB; GRID; HPRD) HCK SOS1(HPRD; IntAct) HCK STAT3 (HPRD) HCK TRPV4 (GRID; HPRD) HCK TSG101 (HPRD;IntAct) HCK UNC119 (CCSB; GRID; HPRD) HCK VAV1 (HPRD) HCK WAS (HPRD;IntAct; MINT) HCK WIPF1 (CCSB; GRID; HPRD; IntAct; MINT) IGF1R ARHGEF12(GRID; HPRD; MINT) IGF1R CRK (HPRD) IGF1R CRKL (HPRD) IGF1R DOK4 (HPRD)IGF1R DOK5 (HPRD) IGF1R EHD1 (BIND; GRID; HPRD) IGF1R ESR1 (HPRD) IGF1RGIGYF1 (GRID; HPRD) IGF1R GIPC1 (HPRD) IGF1R GNAI1 (GRID; HPRD) IGF1RGNAI2 (GRID; HPRD) IGF1R GNB2L1 (BIND; GRID; HPRD) IGF1R GRB10 (BIND;GRID; HPRD; MINT) IGF1R GYS1 (BIND) IGF1R HNF1A (BIND) IGF1R IGF1 (BIND;DIP; GRID; HPRD) IGF1R IGF2 (GRID; HPRD) IGF1R IGFBP3 (GRID; HPRD) IGF1RINS (BIND; GRID; HPRD) IGF1R INSR (HPRD) IGF1R IRS1 (BIND; GRID; HPRD)IGF1R IRS2 (GRID; HPRD; MINT) IGF1R IRS4 (HPRD) IGF1R ITGB1 (BIND; HPRD)IGF1R JAK1 (BIND; GRID; HPRD) IGF1R JAK2 (HPRD) IGF1R KRT27 (HPRD) IGF1RMAP3K5 (HPRD) IGF1R MDM2 (HPRD) IGF1R NEDD4 (GRID; HPRD) IGF1R PBEF1(BIND) IGF1R PIK3R1 (DIP; GRID; HPRD) IGF1R PIK3R2 (BIND; HPRD) IGF1RPIK3R3 (GRID; HPRD) IGF1R PRKCD (BIND; HPRD) IGF1R PRKD1 (BIND; HPRD)IGF1R PTPN1 (GRID; HPRD) IGF1R PTPN11 (DIP; GRID; HPRD) IGF1R RASA1(GRID; HPRD) IGF1R SHC1 (BIND; DIP; GRID; HPRD; IntAct) IGF1R SNAP29(GRID) IGF1R SOCS1 (BIND; GRID; HPRD; MINT) IGF1R SOCS2 (BIND; GRID;HPRD; MINT) IGF1R SOCS3 (BIND; GRID; HPRD; MINT) IGF1R SRC (HPRD) IGF1RSTAT3 (HPRD) IGF1R TAF1 (BIND) IGF1R VAV3 (GRID: HPRD) IGF1R WISP2(HPRD) IGF1R YWHAB (BIND; GRID; HPRD; IntAct; MINT) IGF1R YWHAE (BIND;GRID; HPRD; MINT) IGF1R YWHAG (GRID; HPRD; MINT) IGF1R YWHAZ (BIND;GRID; HPRD) INSR ACP1 (HPRD) INSR ADRB2 (HPRD) INSR AHSG (GRID; HPRD)INSR ARF1 (BIND; HPRD) INSR ARHGAP26 (HPRD) INSR CALM1 (HPRD) INSR CALM2(HPRD) INSR CALM3 (HPRD) INSR CASP7 (BIND) INSR CAV1 (GRID; HPRD) INSRCAV3 (HPRD) INSR CBL (HPRD) INSR CEACAM1 (HPRD) INSR CRK (HPRD) INSRCRKL (HPRD; IntAct) INSR DOK1 (HPRD; reactome) INSR DOK4 (HPRD) INSRDOK5 (HPRD) INSR ENPP1 (GRID; HPRD) INSR FABP4 (HPRD) INSR FRS2 (GRID;HPRD) INSR GAB1 (GRID; HPRD) INSR GNB2L1 (HPRD) INSR GRB10 (BIND; GRID;HPRD; MINT; reactome) INSR GRB14 (GRID; HPRD; MINT) INSR GRB2 (reactome)INSR GRB7 (GRID; HPRD) INSR HMGA1 (BIND) INSR HNF1A (BIND) INSR HRAS(GRID; HPRD) INSR IGF2 (GRID; HPRD) INSR IMPDH2 (BIND) INSR INS (DIP;GRID; HPRD; reactome) INSR INSRR (GRID; HPRD) INSR IRS1 (BIND; DIP;GRID; HPRD; IntAct; MINT; reactome) INSR IRS2 (GRID; HPRD; MINT;reactome) INSR JAK1 (BIND; HPRD) INSR JAK2 (HPRD) INSR KHDRBS1 (HPRD)INSR KRT27 (HPRD) INSR MAD2L1 (HPRD; MINT) INSR MAPK3 (GRID; HPRD) INSRPBEF1 (BIND) INSR PIK3CA (reactome) INSR PIK3CB (reactome) INSR PIK3R1(BIND; DIP; HPRD; reactome) INSR PIK3R2 (reactome) INSR PIK3R3 (GRID;HPRD) INSR PLCG1 (HPRD) INSR PRKCA (HPRD) INSR PRKCD (GRID; HPRD) INSRPTK2 (HPRD) INSR PTPN1 (GRID; HPRD; MINT) INSR PTPN11 (DIP; GRID; HPRD)INSR PTPN12 (GRID; HPRD) INSR PTPN2 (HPRD) INSR PTPN6 (GRID; HPRD) INSRPTPRC (HPRD) INSR PTPRF (GRID; HPRD) INSR RAF1 (GRID; HPRD) INSR RASA1(GRID; HPRD) INSR SH2B1 (GRID; HPRD) INSR SH2B2 (HPRD) INSR SHC1 (BIND;DIP; GRID; HPRD; IntAct; reactome) INSR SMAD2 (GRID; HPRD) INSR SNX1(GRID; HPRD) INSR SNX2 (GRID; HPRD) INSR SNX4 (GRID; HPRD) INSR SNX6(GRID; HPRD) INSR SOCS1 (BIND; HPRD) INSR SOCS2 (HPRD) INSR SOCS3 (GRID;HPRD) INSR SOCS6 (BIND; HPRD) INSR SORBS1 (GRID; HPRD) INSR SOS1(reactome) INSR SRC (HPRD) INSR STAT5A (HPRD) INSR STAT5B (HPRD) INSRSYNCRIP (GRID; HPRD) INSR VAV1 (GRID; HPRD) INSR VAV3 (GRID; HPRD) INSRYWHAB (HPRD; IntAct) INSRR KRT27 (HPRD) ITK BLNK (GRID; HPRD) ITK CBL(GRID; HPRD) ITK CD28 (GRID; HPRD) ITK GRB2 (GRID; HPRD) ITK HNRPK(BIND; GRID; HPRD) ITK KHDRBS1 (GRID; HPRD) ITK KPNA2 (BIND; GRID; HPRD)ITK LAT (BIND; GRID; HPRD) ITK LCK (HPRD) ITK LCP2 (BIND; GRID; HPRD;MINT) ITK PLCG1 (BIND; GRID; HPRD) ITK PLCG2 (HPRD) ITK PPIA (GRID;HPRD) ITK SH2D2A (HPRD) ITK SMAD4 (BIND; HPRD) ITK SOCS1 (BIND; GRID;HPRD) ITK TGFBR1 (BIND; HPRD) ITK WAS (GRID; HPRD) JAK1 ARRB1 (GRID)JAK1 ATIC (GRID) JAK1 BRCA1 (GRID; HPRD; MINT) JAK1 CCR1 (HPRD) JAK1CCR5 (HPRD) JAK1 CSF2RB (HPRD) JAK1 CSF3R (GRID; HPRD) JAK1 CXCR4 (HPRD)JAK1 ELF3 (GRID) JAK1 ELP2 (GRID HPRD) JAK1 GHR (GRID, HPRD) JAK1 GNB2L1(BIND, GRID HPRD, MINT) JAK1 GRB2 (DIP, GRID, HPRD) JAK1 IFNAR1 (DIP)JAK1 IFNAR2 (BIND, HPRD, MINT) JAK1 IFNGR1 (GRID, HPRD) JAK1 IL10RA(BIND, GRID, HPRD) JAK1 IL21R (GRID, HPRD) JAK1 IL2RB (BIND DIP, GRIDHPRD) JAK1 IL2RG (HPRD) JAK1 IL3RA (HPRD) JAK1 IL4R (BIND, GRID HPRD)JAK1 IL5RA (GRID, HPRD) JAK1 IL6R (MINT) JAK1 IL6ST (DIP, GRID, HPRD)JAK1 IL7R (HPRD) JAK1 IL9R (GRID, HPRD) JAK1 INPP5D (HPRD) JAK1 IRS1(HPRD) JAK1 IRS2 (GRID HPRD) JAK1 JAK3 (GRID, HPRD) JAK1 JAKMIP1 (BINDHPRD) JAK1 LRPPRC (MINT) JAK1 MDK (GRID, HPRD) JAK1 OSMR (GRID, HPRD)JAK1 PDGFRA (HPRD) JAK1 PDGFRB (HPRD) JAK1 PIK3R1 (GRID, HPRD) JAK1PLA2G4A (HPRD) JAK1 PLAUR (BIND, DIP, GRID, HPRD) JAK1 PLCG2 (HPRD) JAK1PRKCZ (HPRD) JAK1 PRMT5 (GRID, HPRD) JAK1 PTPN11 (GRID, HPRD) JAK1 PTPN2(HPRD) JAK1 PTPN6 (HPRD) JAK1 PTPRC (HPRD) JAK1 RAF1 (GRID HPRD) JAK1SH2B2 (HPRD) JAK1 SHB (GRID, HPRD) JAK1 SOCS1 (GRID HPRD) JAK1 SOCS3(HPRD) JAK1 STAM (HPRD) JAK1 STAM2 (GRID, HPRD, MINT) JAK1 STAT1 (GRID,HPRD) JAK1 STAT2 (HPRD) JAK1 STAT3 (GRID, HPRD, MINT) JAK1 STAT5A (GRIDHPRD) JAK1 STAT6 (HPRD) JAK1 SYK (GRID, HPRD) JAK1 TAF1 (BIND) JAK1 TEC(GRID, HPRD) JAK1 TNFRSF1A (GRID, HPRD) JAK1 TSHR (GRID, HPRD) JAK1 TYK2(GRID, HPRD) JAK2 AGTR1 (GRID, HPRD) JAK2 BCR (GRID, HPRD) JAK2 BRCA1(GRID, HPRD, MINT) JAK2 CCR2 (GRID, HPRD) JAK2 CCR5 (GRID, HPRD) JAK2CRLF2 (HPRD) JAK2 CSF2RB (GRID, HPRD) JAK2 CSF3R (GRID, HPRD) JAK2 CTLA4(BIND, GRID, HPRD) JAK2 CXCR4 (GRID, HPRD) JAK2 DNAJA3 (BIND, GRID,HPRD) JAK2 ELP2 (GRID, HPRD) JAK2 EPOR (GRID; HPRD) JAK2 GHR (GRID;HPRD) JAK2 GRB10 (GRID; HPRD) JAK2 GRB2 (GRID; HPRD) JAK2 GTF2I (HPRD)JAK2 HES1 (HPRD) JAK2 HES5 (HPRD) JAK2 HSPA8 (GRID; HPRD) JAK2 HTR2A(GRID; HPRD) JAK2 IFNGR1 (HPRD) JAK2 IFNGR2 (GRID; HPRD) JAK2 IL12RB2(BIND; GRID; HPRD) JAK2 IL23R (HPRD) JAK2 IL3RA (HPRD) JAK2 IL4R (HPRD)JAK2 IL5RA (GRID; HPRD) JAK2 IRS1 (HPRD) JAK2 IRS2 (HPRD) JAK2 JAK3(HPRD) JAK2 KIT (GRID; HPRD) JAK2 LEPR (GRID; HPRD; IntAct) JAK2 LYN(GRID; HPRD) JAK2 MDK (GRID; HPRD) JAK2 MPL (HPRD) JAK2 MST1R (HPRD)JAK2 NFKBIA (HPRD) JAK2 OSMR (GRID; HPRD) JAK2 PDGFRB (HPRD) JAK2 PIK3R1(GRID; HPRD) JAK2 PKD1 (HPRD) JAK2 PLCG2 (HPRD) JAK2 PPIA (GRID; HPRD)JAK2 PPP2CA (HPRD) JAK2 PPP2R1B (GRID; HPRD) JAK2 PPP2R5A (GRID; HPRD)JAK2 PRLR (GRID; HPRD) JAK2 PRMT5 (GRID; HPRD) JAK2 PTK2 (GRID; HPRD)JAK2 PTPN1 (GRID; HPRD) JAK2 PTPN11 (GRID; HPRD) JAK2 PTPN12 (GRID;HPRD) JAK2 PTPN6 (GRID; HPRD) JAK2 PTPRC (HPRD) JAK2 RAF1 (GRID; HPRD)JAK2 SH2B1 (GRID; HPRD) JAK2 SH2B2 (HPRD) JAK2 SHC1 (GRID; HPRD) JAK2SIRPA (GRID; HPRD) JAK2 SOCS1 (GRID; HPRD) JAK2 SOCS3 (GRID; HPRD) JAK2STAM (GRID; HPRD) JAK2 STAM2 (GRID; HPRD) JAK2 STAP2 (HPRD) JAK2 STAT1(HPRD) JAK2 STAT2 (HPRD) JAK2 STAT3 (GRID; HPRD) JAK2 STAT5A (GRID;HPRD) JAK2 STAT5B (HPRD) JAK2 TEC (GRID; HPRD) JAK2 TNFRSF1A (GRID;HPRD) JAK2 TSHR (GRID; HPRD) JAK2 TUB (GRID; HPRD) JAK2 TYK2 (HPRD) JAK2UBASH3B (HPRD) JAK2 VAV1 (GRID; HPRD) JAK2 VCP (GRID; HPRD) JAK2 YES1(GRID; HPRD) JAK3 CD247 (CCSB; GRID; HPRD) JAK3 CD40 (HPRD) JAK3 CXCR4(CCSB; GRID; HPRD) JAK3 IL2RB (CCSB; GRID; HPRD) JAK3 IL2RG (CCSB; DIP;GRID; HPRD) JAK3 IL4R (GRID; HPRD) JAK3 IL6ST (HPRD) JAK3 IL7R (HPRD)JAK3 INPP5D (HPRD) JAK3 IRS1 (GRID; HPRD) JAK3 IRS2 (GRID; HPRD) JAK3KHDRBS1 (HPRD; IntAct) JAK3 LCK (CCSB; GRID; HPRD) JAK3 PIK3R1 (HPRD)JAK3 PRMT5 (GRID; HPRD) JAK3 PTPN2 (HPRD) JAK3 PTPN6 (HPRD) JAK3 PTPRC(GRID; HPRD) JAK3 SH2B2 (HPRD) JAK3 SHB (GRID; HPRD) JAK3 SOCS1 (GRID;HPRD) JAK3 SOCS3 (HPRD) JAK3 STAM (CCSB; GRID; HPRD) JAK3 STAM2 (CCSB;GRID; HPRD) JAK3 STAT3 (CCSB; GRID; HPRD) JAK3 STAT5A (CCSB; GRID; HPRD)JAK3 TIAF1 (GRID; HPRD) KDR ACP1 (GRID; HPRD) KDR ANXA5 (GRID; HPRD) KDRATR (HPRD) KDR CAV1 (HPRD) KDR CBL (HPRD) KDR CDH5 (GRID; HPRD; MINT)KDR COL18A1 (GRID; HPRD) KDR CRK (HPRD) KDR CSF2RB (HPRD) KDR CTNNB1(HPRD) KDR DNM2 (HPRD) KDR FIGF (DIP; GRID; HPRD) KDR FRS2 (HPRD) KDRGNA11 (HPRD) KDR GNAQ (HPRD) KDR GRB10 (GRID; HPRD) KDR GRB2 (GRID;HPRD) KDR HSP90AA1 (HPRD) KDR IQGAP1 (HPRD) KDR ITGB3 (HPRD) KDR NCK1(HPRD; MINT) KDR NRP1 (HPRD; MINT) KDR P2RY2 (HPRD) KDR PLCG1 (BIND;HPRD) KDR PLCG2 (GRID; HPRD) KDR PLXNA1 (GRID) KDR PTPN11 (HPRD) KDRPTPN6 (HPRD) KDR RASA1 (HPRD) KDR SH2D2A (BIND; HPRD) KDR SHB (GRID;HPRD) KDR SHC1 (GRID; HPRD; MINT) KDR SHC2 (BIND; GRID; HPRD; MINT) KDRSRC (HPRD) KDR STAT1 (HPRD) KDR SYNGAP1 (HPRD) KDR TIMP3 (HPRD) KDRVEGFA (BIND; DIP; GRID; HPRD; MINT) KDR VEGFC (BIND; GRID; HPRD) KDRYES1 (HPRD) KIT BCR (GRID) KIT CBL (HPRD) KIT CBLB (HPRD) KIT CD63(GRID) KIT CD81 (GRID; HPRD) KIT CD9 (GRID; HPRD) KIT CISH (HPRD) KITCLTC (GRID; HPRD) KIT CRK (HPRD) KIT CRKL (GRID; HPRD) KIT CSF2RA (HPRD)KIT CSF2RB (HPRD) KIT DOK1 (GRID; HPRD) KIT EPOR (GRID; HPRD) KIT GRAP(GRID; HPRD) KIT GRAP2 (GRID; HPRD) KIT GRB10 (GRID; HPRD) KIT GRB2(BIND; DIP; GRID; HPRD) KIT GRB7 (GRID; HPRD) KIT INPP5D (HPRD) KITKITLG (GRID; HPRD) KIT LCK (GRID; HPRD) KIT LYN (BIND; GRID; HPRD) KITMATK (GRID; HPRD) KIT MPDZ (GRID; HPRD) KIT PIK3CG (HPRD) KIT PIK3R1(BIND; GRID; HPRD) KIT PIK3R2 (BIND; GRID; HPRD) KIT PLCE1 (HPRD) KITPLCG1 (BIND; GRID; HPRD) KIT PRKCA (HPRD) KIT PRKCB1 (HPRD) KIT PTPN11(BIND; GRID; HPRD) KIT PTPN6 (GRID; HPRD) KIT PTPRO (GRID; HPRD) KITPTPRU (HPRD) KIT RASA1 (HPRD) KIT SH2B2 (HPRD) KIT SH2B3 (HPRD) KITSOCS1 (BIND; GRID; HPRD) KIT SOCS5 (HPRD) KIT SOCS6 (HPRD) KIT SPRED1(HPRD) KIT SPRED2 (HPRD) KIT SRC (HPRD) KIT STAP1 (HPRD) KIT STAT1(BIND; GRID; HPRD) KIT STAT5A (HPRD) KIT STAT5B (HPRD) KIT TEC (GRID;HPRD) KIT YES1 (HPRD) LCK ACP1 (HPRD) LCK ADAM15 (CCSB; GRID; HPRD;MINT) LCK AP2A1 (reactome) LCK AP2A2 (reactome) LCK AP2B1 (reactome) LCKAP2M1 (reactome) LCK AP2S1 (reactome) LCK ATP6V1H (reactome) LCK BCAR1(GRID; HPRD) LCK CAMLG (BIND) LCK CBL (GRID; HPRD; MINT) LCK CCR5 (GRID;HPRD) LCK CD2 (CCSB; GRID; HPRD; MINT) LCK CD247 (HPRD; MINT) LCK CD28(GRID; HPRD) LCK CD38 (BIND; CCSB; GRID; HPRD) LCK CD3E (GRID; HPRD) LCKCD4 (CCSB; GRID; HPRD; MINT; reactome) LCK CD44 (CCSB; GRID; HPRD) LCKCD48 (CCSB; GRID; HPRD) LCK CD5 (DIP; GRID; HPRD) LCK CD55 (CCSB; GRID;HPRD) LCK CD79A (HPRD) LCK CD79B (HPRD) LCK CD8A (HPRD) LCK CD8B (BIND)LCK CDC25C (CCSB; GRID; HPRD) LCK CSF2RB (HPRD) LCK CSF3R (HPRD) LCKCTLA4 (GRID; HPRD) LCK CTNND1 (GRID) LCK CTNND2 (GRID; HPRD) LCK DAPP1(HPRD) LCK DEF6 (HPRD) LCK DLG1 (GRID; HPRD) LCK DNM2 (BIND) LCK DOK1(HPRD) LCK DOK2 (CCSB; GRID; HPRD) LCK DOK3 (HPRD) LCK ESR1 (HPRD) LCKESR2 (HPRD) LCK EZR (HPRD) LCK FAS (CCSB; GRID; HPRD) LCK FASLG (GRID;HPRD) LCK FCGR3A (CCSB; GRID; HPRD) LCK GAB2 (HPRD) LCK GRAP (HPRD) LCKHSP90AA1 (DIP; MINT) LCK IFNAR1 (HPRD) LCK IL2RB (CCSB; GRID; HPRD) LCKKHDRBS1 (BIND; GRID; HPRD; IntAct; MINT) LCK KIR2DL3 (HPRD) LCK LAT(HPRD) LCK LAX1 (HPRD) LCK LCP2 (HPRD; MINT) LCK LIME1 (HPRD) LCK LYN(HPRD) LCK MAPK1 (HPRD; MINT) LCK MAPK3 (HPRD) LCK MS4A1 (HPRD) LCK MUC1(HPRD) LCK NEDD9 (GRID; HPRD) LCK NFKBIA (BIND; HPRD) LCK NOTCH1 (GRID;HPRD) LCK NR3C1 (MINT) LCK NXF1 (IntAct) LCK PAG1 (GRID; HPRD) LCKPECAM1 (GRID; HPRD) LCK PIK3CA (GRID; HPRD) LCK PIK3R1 (GRID; HPRD;MINT) LCK PLCG1 (GRID; HPRD) LCK PLCG2 (HPRD) LCK PLD2 (GRID; HPRD) LCKPRKACA (HPRD) LCK PRKCA (HPRD) LCK PRKCD (HPRD) LCK PRKCQ (HPRD) LCKPTK2 (HPRD) LCK PTPN11 (CCSB; GRID; HPRD) LCK PTPN6 (CCSB; GRID; HPRD)LCK PTPRC (GRID; HPRD; IntAct) LCK PTPRF (HPRD) LCK PTPRH (HPRD) LCK PXN(GRID; HPRD) LCK RAF1 (CCSB; GRID; HPRD) LCK RASA1 (HPRD) LCK SH2B3(CCSB; GRID; HPRD) LCK SH2D1A (HPRD; MINT) LCK SH2D2A (HPRD; IntAct) LCKSH3BP2 (HPRD) LCK SHC1 (HPRD) LCK SIT1 (GRID; HPRD) LCK SKAP1 (GRID;HPRD) LCK SQSTM1 (CCSB; GRID; HPRD) LCK STAT1 (HPRD) LCK STAT3 (CCSB;GRID; HPRD) LCK STAT5A (HPRD) LCK SYK (CCSB; GRID; HPRD; MINT) LCK TEK(GRID; HPRD) LCK THY1 (CCSB; GRID; HPRD) LCK TRAT1 (HPRD) LCK TRPV4(GRID; HPRD) LCK TUB (GRID; HPRD) LCK UNC119 (CCSB; GRID; HPRD) LCK VAV1(HPRD) LCK WASL (IntAct) LCK ZAP70 (CCSB; GRID; HPRD; MINT) LMTK2 CDK5(GRID) LMTK2 CDK5R1 (GRID; HPRD) LMTK2 PPP1R2 (GRID; HPRD) LMTK3 ZBTB16(MDC) LTK CBL (GRID; HPRD) LTK PIK3C2B (GRID; HPRD) LTK PLCG1 (HPRD) LTKPTPN1 (GRID; HPRD) LTK SHC1 (GRID; HPRD) LYN ACTB (HPRD) LYN ADAM15(MINT) LYN BANK1 (GRID; HPRD) LYN BCAR1 (GRID; HPRD) LYN CASP3 (HPRD)LYN CASP7 (HPRD) LYN CASP9 (HPRD) LYN CBL (BIND; DIP; GRID; HPRD; MINT)LYN CBLC (GRID; HPRD) LYN CD19 (GRID; HPRD) LYN CD22 (GRID; HPRD;IntAct) LYN CD24 (GRID; HPRD) LYN CD36 (GRID; HPRD) LYN CD79A (HPRD) LYNCD79B (HPRD) LYN CDAN3 (GRID) LYN CDC2 (GRID; HPRD) LYN CDK2 (GRID;HPRD) LYN CDK4 (GRID) LYN CDKN1B (HPRD) LYN CRKL (HPRD) LYN CSF2RA(HPRD) LYN CSF2RB (GRID; HPRD) LYN CSF3R (GRID; HPRD) LYN CSNK2B (BIND;GRID; HPRD; IntAct; MINT) LYN CTLA4 (GRID; HPRD) LYN DAPP1 (HPRD) LYNDLG4 (GRID; HPRD) LYN DOK1 (HPRD) LYN DOK3 (HPRD) LYN EPOR (GRID; HPRD)LYN EVL (GRID; HPRD) LYN FCAR (GRID; HPRD) LYN FCER1G (GRID; HPRD) LYNFCGR2A (HPRD) LYN FCGR2B (HPRD) LYN FOLR1 (GRID; HPRD) LYN GAB2 (GRID;HPRD) LYN GAB3 (GRID; HPRD) LYN GALNAC4S-6ST (GRID; HPRD) LYN GP6 (GRID;HPRD) LYN GRIA3 (GRID; HPRD) LYN HCLS1 (GRID; HPRD; MINT) LYN HNRPK(HPRD) LYN IL2RB (HPRD) LYN IL7R (GRID; HPRD) LYN INPP5D (GRID; HPRD)LYN ITPR1 (HPRD) LYN KHDRBS1 (HPRD; IntAct; MINT) LYN LCP2 (BIND; GRID;HPRD) LYN LIME1 (HPRD) LYN MAP3K3 (IntAct; MINT) LYN MAP4K1 (HPRD) LYNMAPK3 (GRID; HPRD) LYN MATK (HPRD) LYN MME (GRID; HPRD) LYN MS4A1 (HPRD)LYN MS4A2 (GRID; HPRD) LYN MUC1 (GRID) LYN NEDD9 (GRID; HPRD) LYN NMT1(GRID; HPRD) LYN NPHS1 (GRID; HPRD) LYN PAG1 (GRID; HPRD) LYN PDE4A(GRID; HPRD) LYN PDE4D (BIND) LYN PECAM1 (GRID; HPRD) LYN PIK3CG (GRID;HPRD) LYN PILRB (HPRD) LYN PLAUR (DIP) LYN PLCG1 (GRID; HPRD) LYN PLCG2(HPRD) LYN PPP1R15A (BIND; GRID; HPRD) LYN PPP1R8 (GRID; HPRD) LYN PRAM1(GRID; HPRD) LYN PRKCD (HPRD) LYN PRKCQ (GRID; HPRD) LYN PRKDC (GRID;HPRD) LYN PTK2 (HPRD; MINT) LYN PTPN6 (GRID; HPRD) LYN PTPRC (HPRD) LYNRASA1 (HPRD) LYN RGS16 (BIND; HPRD) LYN SH2B2 (HPRD) LYN SHC1 (GRID;HPRD; MINT) LYN SKAP1 (GRID; HPRD) LYN SKAP2 (GRID; HPRD) LYN SLC4A1(HPRD) LYN SNCA (HPRD) LYN SPHK2 (IntAct) LYN SYK (DIP; GRID; HPRD;MINT) LYN TEC (HPRD) LYN TEK (HPRD) LYN TRAT1 (HPRD) LYN TRPV4 (GRID;HPRD) LYN TYK2 (GRID; HPRD) LYN UBB (BIND; HPRD) LYN UNC119 (GRID; HPRD)LYN YES1 (HPRD) MATK CD36 (GRID; HPRD) MATK EWSR1 (CCSB; GRID; HPRD;IntAct) MATK NTRK1 (GRID; HPRD) MATK PXN (GRID; HPRD) MATK SRC (CCSB;GRID; HPRD) MERTK BMPR2 (HPRD) MERTK GAS6 (GRID; HPRD; reactome) MERTKGRB2 (GRID; HPRD) MERTK LMO4 (BIND; HPRD) MERTK PROS1 (reactome) MERTKVAV1 (HPRD) MET BAG1 (GRID; HPRD) MET CASP3 (HPRD) MET CBL (BIND; GRID;HPRD; MINT) MET CDH1 (GRID) MET CNR1 (HPRD) MET CTNNB1 (GRID; HPRD) METCTTN (HPRD) MET DAPK3 (HPRD) MET DNAJA3 (HPRD) MET FAS (HPRD) MET GAB1(BIND; GRID; HPRD; MINT) MET GLMN (GRID; HPRD) MET GRB2 (GRID; HPRD) METHGF (BIND; GRID; HPRD; IntAct) MET HGFAC (DIP) MET HGS (GRID; HPRD) METINPP5D (BIND; HPRD) MET INPPL1 (BIND; HPRD) MET ITGB4 (HPRD) MET MUC20(HPRD) MET PCBD2 (HPRD) MET PIK3R1 (BIND; HPRD) MET PLCG1 (HPRD) METPLXNB1 (HPRD; MINT) MET POLR2A (BIND) MET PTPN11 (GRID; HPRD) MET PTPRB(HPRD) MET PTPRJ (HPRD) MET RANBP10 (GRID; HPRD) MET RANBP9 (GRID; HPRD;MINT) MET SH3KBP1 (GRID) MET SHC1 (GRID; HPRD) MET SMC1A (HPRD) METSNAPIN (HPRD) MET SNX2 (HPRD) MET SPSB1 (HPRD) MET SRC (GRID; HPRD) METSTAT3 (GRID; HPRD) MET TAF1 (BIND) MET VAV1 (HPRD) MST1R AKT1 (HPRD)MST1R EPOR (HPRD) MST1R GAB1 (HPRD) MST1R GRB2 (GRID; HPRD) MST1R HYAL2(GRID; HPRD) MST1R MAX (BIND) MST1R MST1 (DIP; GRID; HPRD) MST1R MYC(BIND) MST1R PIK3R1 (GRID; HPRD) MST1R PLCG1 (GRID; HPRD) MST1R RELA(HPRD) MST1R SFN (HPRD) MST1R SHC1 (GRID; HPRD) MST1R SRC (GRID; HPRD)MST1R YES1 (HPRD) MST1R YWHAB (HPRD) MST1R YWHAE (HPRD) MST1R YWHAH(HPRD) MST1R YWHAQ (HPRD) MST1R YWHAZ (HPRD) MUSK COLQ (HPRD) MUSK DOK7(HPRD) MUSK RAPSN (GRID; HPRD) MUSK SYNE1 (GRID; HPRD) NTRK1 ADCYAP1(reactome) NTRK1 ADCYAP1R1 (reactome) NTRK1 ADORA2A (reactome) NTRK1BRAF (reactome) NTRK1 CAV1 (GRID; HPRD) NTRK1 CCNA2 (BIND) NTRK1 CPSF4(BIND) NTRK1 CRK (HPRD; reactome) NTRK1 DNAJA3 (HPRD) NTRK1 DYNLL1(HPRD) NTRK1 FRS2 (BIND; GRID; HPRD; reactome) NTRK1 FRS3 (BIND; GRID;HPRD) NTRK1 GIPC1 (BIND; GRID; HPRD) NTRK1 GRB2 (GRID; HPRD) NTRK1HIST3H3 (BIND) NTRK1 HIST4H4 (BIND) NTRK1 HRAS (reactome) NTRK1 IRS1(HPRD) NTRK1 IRS2 (HPRD) NTRK1 KIDINS220 (HPRD; reactome) NTRK1 KRAS(reactome) NTRK1 MAPK3 (HPRD) NTRK1 NEDD4L (HPRD) NTRK1 NGF (BIND; DIP;GRID; HPRD; reactome) NTRK1 NGFR (DIP; GRID; HPRD) NTRK1 NRAS (reactome)NTRK1 PIK3R1 (HPRD) NTRK1 PLCG1 (GRID; HPRD; reactome) NTRK1 PTPN1(GRID; HPRD) NTRK1 PTPN11 (HPRD) NTRK1 RAF1 (reactome) NTRK1 RAP1A(GRID; HPRD; reactome) NTRK1 RAPGEF1 (reactome) NTRK1 RASA1 (HPRD) NTRK1RICS (GRID; HPRD) NTRK1 RIT1 (reactome) NTRK1 RIT2 (reactome) NTRK1RUSC1 (HPRD) NTRK1 SH2B1 (HPRD) NTRK1 SH2B2 (HPRD) NTRK1 SHC1 (BIND;HPRD; reactome) NTRK1 SHC2 (HPRD; reactome) NTRK1 SHC3 (GRID; HPRD;reactome) NTRK1 SQSTM1 (HPRD) NTRK1 STAT3 (reactome) NTRK1 UBB (HPRD)NTRK1 YWHAB (reactome) NTRK2 ADCYAP1 (reactome) NTRK2 ADCYAP1R1(reactome) NTRK2 ADORA2A (reactome) NTRK2 ATP5O (BIND) NTRK2 BDNF (DIP;GRID; HPRD) NTRK2 DOK5 (HPRD) NTRK2 DYNLL1 (GRID; HPRD) NTRK2 FRS3(GRID; HPRD) NTRK2 GIPC1 (GRID; HPRD) NTRK2 KIDINS220 (HPRD) NTRK2 NCK1(GRID; HPRD) NTRK2 NCK2 (GRID; HPRD) NTRK2 NGFR (HPRD) NTRK2 NTF3 (GRID;HPRD) NTRK2 NTF4 (BIND; GRID; HPRD) NTRK2 PLCG1 (HPRD) NTRK2 PTPN1(GRID; HPRD) NTRK2 PTPN11 (HPRD) NTRK2 SH2B1 (HPRD) NTRK2 SH2B2 (HPRD)NTRK2 SH2D1A (MINT) NTRK2 SHC1 (HPRD) NTRK2 SHC2 (HPRD) NTRK2 SHC3(GRID; HPRD) NTRK2 SQSTM1 (GRID; HPRD) NTRK2 TNFRSF1A (BIND) NTRK2 TRAF2(BIND) NTRK2 UBB (HPRD) NTRK3 CYP2D6 (BIND) NTRK3 DOK5 (HPRD) NTRK3DYNLL1 (HPRD) NTRK3 FOS (MINT) NTRK3 HTR2A (HPRD) NTRK3 IRAK1 (GRID;HPRD) NTRK3 JUN (MINT) NTRK3 KIDINS220 (HPRD) NTRK3 MAPK1 (HPRD; MINT)NTRK3 MAPK3 (HPRD) NTRK3 NGFR (GRID; HPRD) NTRK3 NGFRAP1 (CCSB; GRID;HPRD) NTRK3 NTF3 (DIP; GRID; HPRD; MINT) NTRK3 PLCG1 (GRID; HPRD) NTRK3PMAIP1 (BIND) NTRK3 PTPN1 (CCSB; GRID; HPRD) NTRK3 SH2D1A (MINT) NTRK3SHC1 (CCSB; GRID; HPRD) NTRK3 SHC2 (HPRD) NTRK3 SQSTM1 (CCSB; GRID;HPRD) NTRK3 TNFRSF1A (BIND) NTRK3 TRAF2 (BIND) PDGFRA CAV1 (CCSB; GRID;HPRD) PDGFRA CBL (GRID) PDGFRA CRK (CCSB; GRID; HPRD) PDGFRA CRKL (GRID;HPRD) PDGFRA GRB14 (GRID; HPRD) PDGFRA ITGAV (GRID; HPRD) PDGFRA ITGB3(GRID; HPRD) PDGFRA PDGFA (DIP; GRID; HPRD) PDGFRA PDGFB (CCSB; DIP;GRID; HPRD) PDGFRA PDGFC (GRID; HPRD) PDGFRA PDGFRB (CCSB; GRID; HPRD)PDGFRA PIK3R1 (HPRD) PDGFRA PLCG1 (GRID; HPRD) PDGFRA PTPN11 (CCSB;GRID; HPRD) PDGFRA RAPGEF1 (GRID; HPRD) PDGFRA SHB (GRID; HPRD) PDGFRASHF (HPRD) PDGFRA SLC9A3R1 (GRID; HPRD) PDGFRA SNX2 (GRID; HPRD) PDGFRASNX4 (CCSB; GRID; HPRD) PDGFRA SNX6 (GRID; HPRD) PDGFRA STAT1 (HPRD)PDGFRA STAT3 (HPRD) PDGFRA STAT5A (HPRD) PDGFRA STAT5B (HPRD) PDGFRBARAF (HPRD) PDGFRB BAG1 (CCSB; GRID; HPRD) PDGFRB CBL (BIND; HPRD)PDGFRB CBLC (HPRD) PDGFRB COPA (GRID; HPRD) PDGFRB COPB1 (CCSB; GRID;HPRD) PDGFRB CRK (BIND; CCSB; GRID; HPRD) PDGFRB EDG1 (CCSB; GRID; HPRD)PDGFRB EIF2AK2 (GRID; HPRD) PDGFRB GAB1 (HPRD) PDGFRB GRB10 (GRID; HPRD)PDGFRB GRB14 (GRID; HPRD) PDGFRB GRB2 (CCSB; DIP; GRID; HPRD) PDGFRBGRB7 (HPRD) PDGFRB ITGB3 (GRID; HPRD) PDGFRB KRTAP4-12 (CCSB; GRID;HPRD; IntAct) PDGFRB NCK1 (BIND; CCSB; GRID; HPRD) PDGFRB NCK2 (CCSB;GRID; HPRD) PDGFRB PDAP1 (CCSB; GRID; HPRD) PDGFRB PDGFB (CCSB; DIP;GRID; HPRD) PDGFRB PDGFD (HPRD) PDGFRB PIK3C2B (IntAct) PDGFRB PIK3CA(HPRD) PDGFRB PIK3R1 (BIND; DIP; GRID; HPRD) PDGFRB PIK3R2 (BIND; HPRD)PDGFRB PIK3R3 (BIND; CCSB; GRID; HPRD) PDGFRB PLAUR (BIND) PDGFRB PLCG1(BIND; DIP; GRID; HPRD) PDGFRB PRDX2 (HPRD) PDGFRB PTEN (MINT) PDGFRBPTK2 (CCSB; GRID; HPRD) PDGFRB PTPN1 (HPRD) PDGFRB PTPN11 (BIND; CCSB;GRID; HPRD) PDGFRB PTPN2 (HPRD) PDGFRB PTPRJ (HPRD) PDGFRB RAF1 (CCSB;GRID; HPRD) PDGFRB RASA1 (BIND; CCSB; DIP; GRID; HPRD) PDGFRB SH3KBP1(CCSB; GRID) PDGFRB SHB (BIND) PDGFRB SHC1 (BIND; CCSB; GRID; HPRD)PDGFRB SLC9A3R1 (GRID; HPRD; MINT) PDGFRB SLC9A3R2 (MINT) PDGFRB SNX1(CCSB; GRID; HPRD) PDGFRB SNX2 (GRID; HPRD) PDGFRB SNX4 (GRID; HPRD)PDGFRB SOCS1 (BIND; HPRD) PDGFRB SRC (BIND; CCSB; GRID; HPRD) PDGFRBSTAT1 (HPRD) PDGFRB STAT3 (HPRD) PDGFRB STAT5A (DIP; HPRD) PDGFRB STAT5B(HPRD) PDGFRB SYNGAP1 (HPRD) PDGFRB TYK2 (HPRD) PDGFRB YES1 (HPRD) PTK2ACTN1 (HPRD) PTK2 APC (GRID; HPRD) PTK2 ARHGAP26 (GRID; HPRD) PTK2 ATG12(CCSB; GRID; HPRD; MINT) PTK2 BCAR1 (BIND; GRID; HPRD; MINT) PTK2 BIN1(GRID; HPRD) PTK2 BIRC4 (reactome) PTK2 CASP3 (reactome) PTK2 CASP7(reactome) PTK2 CCR5 (GRID; HPRD) PTK2 CD4 (CCSB; GRID) PTK2 CD47 (CCSB;GRID; HPRD) PTK2 CD79B (HPRD) PTK2 CIB1 (GRID; HPRD) PTK2 CRK (GRID;HPRD) PTK2 CRKL (GRID) PTK2 CSPG4 (GRID; HPRD) PTK2 CXCR4 (HPRD) PTK2DCC (BIND; GRID) PTK2 DDEF1 (GRID; HPRD) PTK2 DIABLO (reactome) PTK2DLGAP3 (HPRD) PTK2 DNM2 (BIND; HPRD) PTK2 EFS (CCSB; GRID; HPRD) PTK2EZR (CCSB; GRID; HPRD) PTK2 GIT1 (GRID: HPRD) PTK2 GRB2 (BIND; CCSB;GRID; HPRD) PTK2 GRB7 (CCSB; GRID; HPRD) PTK2 GSK3B (CCSB; GRID; HPRD)PTK2 GZMB (HPRD) PTK2 HNF4A (BIND) PTK2 IGHM (HPRD) PTK2 IRS1 (GRID;HPRD) PTK2 ITGAV (GRID; HPRD) PTK2 ITGB1 (BIND; CCSB; GRID; HPRD) PTK2ITGB2 (CCSB; GRID; HPRD) PTK2 ITGB3 (BIND; GRID; HPRD) PTK2 ITGB4 (HPRD)PTK2 ITGB5 (BIND) PTK2 KCNMA1 (HPRD) PTK2 MAPK8IP3 (GRID; HPRD) PTK2MICAL1 (GRID; HPRD) PTK2 NCAM1 (HPRD) PTK2 NCK2 (CCSB; GRID; HPRD) PTK2NEDD8 (HPRD) PTK2 NEDD9 (GRID; HPRD) PTK2 NEO1 (BIND) PTK2 PIAS1 (HPRD)PTK2 PIK3R1 (GRID; HPRD) PTK2 PKD1 (BIND; GRID; HPRD) PTK2 PLCG1 (GRID;HPRD) PTK2 PPP1CB (CCSB; GRID; HPRD) PTK2 PTEN (GRID) PTK2 PTPN1 (HPRD)PTK2 PTPN11 (CCSB; GRID; HPRD) PTK2 PTPN12 (GRID; HPRD) PTK2 PTPRH(GRID; HPRD) PTK2 PXN (BIND; GRID; HPRD; MINT) PTK2 RB1CC1 (GRID; HPRD)PTK2 RET (HPRD) PTK2 RIPK1 (BIND; HPRD; MINT) PTK2 ROCK1 (HPRD) PTK2SELE (GRID; HPRD) PTK2 SHC1 (BIND; CCSB; GRID; HPRD) PTK2 SOCS2 (CCSB;GRID; HPRD; MINT) PTK2 SORBS1 (HPRD) PTK2 SRC (BIND; CCSB; GRID; HPRD;IntAct; MINT) PTK2 STAT1 (CCSB; GRID; HPRD) PTK2 SYK (CCSB; GRID; HPRD)PTK2 TGFB1I1 (CCSB; GRID; HPRD) PTK2 TLN1 (GRID; HPRD) PTK2 TNFRSF1A(HPRD) PTK2 TNS1 (HPRD) PTK2 TP53 (HPRD) PTK2 TRIO (HPRD) PTK2 TRIP6(CCSB; GRID; HPRD) PTK2 VCL (BIND; HPRD) PTK2B ANXA6 (HPRD) PTK2BARHGAP21 (HPRD) PTK2B ARHGAP5 (HPRD) PTK2B BCAR1 (GRID; HPRD) PTK2B CBL(GRID; HPRD) PTK2B CCR5 (GRID; HPRD) PTK2B CD2AP (GRID) PTK2B CRK (GRID;HPRD) PTK2B DCC (GRID) PTK2B DDEF1 (HPRD) PTK2B DDEF2 (GRID; HPRD) PTK2BDLG3 (GRID; HPRD) PTK2B DLG4 (GRID; HPRD) PTK2B DLGAP3 (HPRD) PTK2B EFS(GRID; HPRD) PTK2B EWSR1 (GRID; HPRD) PTK2B FGFR2 (HPRD) PTK2B FGFR3(HPRD) PTK2B FLT1 (HPRD) PTK2B FYN (GRID; HPRD) PTK2B GNA13 (GRID; HPRD)PTK2B GRB2 (GRID; HPRD) PTK2B GRIN2A (GRID; HPRD) PTK2B GSN (GRID; HPRD)PTK2B IL7R (HPRD) PTK2B ITGB2 (GRID; HPRD) PTK2B ITGB3 (GRID; HPRD)PTK2B JAK1 (GRID; HPRD) PTK2B JAK2 (GRID; HPRD) PTK2B JAK3 (GRID; HPRD)PTK2B KCNA2 (GRID; HPRD) PTK2B LCK (GRID; HPRD) PTK2B LPXN (GRID; HPRD;MINT) PTK2B LYN (GRID; HPRD) PTK2B MAP3K4 (HPRD; MINT) PTK2B MATK (GRID;HPRD) PTK2B MCAM (GRID; HPRD) PTK2B NEDD9 (GRID; HPRD) PTK2B NPHP1(GRID; HPRD) PTK2B PDPK1 (HPRD) PTK2B PIK3R1 (GRID; HPRD) PTK2B PITPNM1(GRID; HPRD) PTK2B PITPNM2 (GRID; HPRD) PTK2B PITPNM3 (GRID; HPRD) PTK2BPRKCD (GRID; HPRD) PTK2B PTK2 (HPRD) PTK2B PTPN11 (GRID; HPRD) PTK2BPTPN12 (GRID; HPRD) PTK2B PTPN6 (GRID; HPRD) PTK2B PXN (GRID; HPRD)PTK2B RASA1 (GRID; HPRD) PTK2B RB1CC1 (GRID; HPRD) PTK2B SH2D3C (HPRD)PTK2B SHC1 (GRID; HPRD) PTK2B SLC2A1 (GRID; HPRD) PTK2B SNCA (HPRD)PTK2B SORBS1 (GRID) PTK2B SORBS2 (BIND; GRID; HPRD) PTK2B SRC (GRID;HPRD) PTK2B STAP1 (HPRD) PTK2B STAT3 (HPRD) PTK2B SYK (GRID; HPRD) PTK2BTGFB1I1 (BIND; GRID; HPRD) PTK2B TLN1 (GRID; HPRD) PTK2B VAV1 (GRID;HPRD) PTK2B ZAP70 (GRID; HPRD) PTK6 IRS1 (BIND) PTK6 IRS4 (BIND) PTK6KHDRBS1 (GRID; HPRD) PTK6 STAP2 (GRID; HPRD) PTK7 E2F4 (BIND) PTK7 HNF4A(BIND) RET AKAP5 (HPRD) RET CBL (HPRD; MINT) RET CBLB (HPRD) RET CRK(HPRD) RET DOK1 (GRID; HPRD) RET DOK2 (CCSB; GRID; HPRD) RET DOK3 (HPRD)RET DOK4 (GRID; HPRD) RET DOK5 (GRID; HPRD) RET DOK6 (GRID; HPRD) RETFAU (MINT) RET FRS2 (BIND; CCSB; GRID; HPRD) RET GAB1 (GRID; HPRD) RETGDNF (GRID; HPRD) RET GFRA1 (CCSB; GRID; HPRD) RET GFRA4 (HPRD) RETGRB10 (GRID; HPRD) RET GRB2 (BIND; CCSB; GRID; HPRD) RET GRB7 (CCSB;GRID; HPRD) RET HIST2H4A (HPRD) RET JUN (BIND) RET MAPK1 (HPRD) RETMAPK14 (HPRD) RET MAPK3 (HPRD) RET MAPK8 (HPRD) RET MAPK9 (HPRD) RETNRTN (GRID; HPRD) RET PDLIM7 (GRID; HPRD) RET PIK3R1 (GRID; HPRD) RETPLCG1 (BIND; GRID; HPRD) RET PRKAR2A (HPRD) RET PTPN11 (HPRD) RET PTPRF(GRID) RET SHC1 (BIND; CCSB; GRID; HPRD; MINT) RET SHC3 (BIND; HPRD) RETSRC (CCSB; GRID; HPRD) RET STAT3 (CCSB; GRID; HPRD) ROR1 NGF (DIP) ROR2FZD2 (HPRD) ROR2 FZD5 (HPRD) ROR2 MAGED1 (GRID; HPRD) ROR2 WNT5A (HPRD)ROS1 LPHN1 (HPRD) ROS1 PTPN6 (GRID; HPRD) ROS1 VAV3 (GRID; HPRD) RYKFZD8 (BIND; HPRD) RYK WNT1 (BIND; HPRD) RYK WNT3A (BIND; HPRD) SRC ACTN1(IntAct) SRC ADAM12 (GRID; HPRD) SRC ADAM15 (CCSB; GRID; HPRD; MINT) SRCADRB2 (HPRD) SRC ADRB3 (GRID; HPRD) SRC ADRBK1 (CCSB; GRID; HPRD) SRCAFAP1 (CCSB; GRID; HPRD) SRC AFAP1L2 (HPRD) SRC AKT1 (HPRD) SRC ANKRD11(GRID; HPRD) SRC ANXA1 (HPRD) SRC ANXA2 (CCSB; GRID; HPRD) SRC AR (GRID)SRC ARHGAP1 (CCSB; GRID; HPRD) SRC ARR3 (CCSB; GRID; HPRD) SRC ARRB1(MINT) SRC ATP2B4 (HPRD) SRC BCAR1 (BIND; GRID; HPRD) SRC BCR (BIND;HPRD) SRC CAV1 (GRID; HPRD; MINT) SRC CAV2 (HPRD) SRC CBL (BIND; GRID;HPRD; MINT) SRC CBLC (HPRD) SRC CCNA2 (DIP) SRC CCND1 (DIP) SRC CD2AP(GRID; HPRD; MINT) SRC CD33 (HPRD) SRC CD36 (GRID; HPRD) SRC CD46 (CCSB;GRID; HPRD) SRC CD59 (CCSB; GRID; HPRD) SRC CDCP1 (BIND; HPRD) SRC CDH1(GRID) SRC CDH5 (HPRD) SRC CDK5 (HPRD) SRC CEACAM1 (HPRD) SRC CEACAM3(HPRD) SRC CHUK (HPRD) SRC CNTNAP1 (HPRD) SRC COL1A1 (reactome) SRCCOL1A2 (reactome) SRC CTNNB1 (HPRD) SRC CTNND1 (HPRD) SRC CTTN (HPRD)SRC DAB1 (HPRD) SRC DAB2 (CCSB; GRID; HPRD) SRC DAG1 (HPRD) SRC DAPP1(HPRD) SRC DDEF1 (GRID; HPRD) SRC DGKA (HPRD) SRC DGKZ (HPRD) SRC DNM1(HPRD; MINT) SRC DNM2 (MINT) SRC DOK1 (HPRD) SRC DOK4 (BIND; HPRD) SRCDRD4 (BIND) SRC E2F4 (BIND) SRC EFNB1 (CCSB; GRID; HPRD) SRC EFNB2(HPRD) SRC EGF (reactome) SRC EPS8 (CCSB; GRID; HPRD) SRC ESR1 (GRID;HPRD) SRC ESR2 (GRID; HPRD) SRC ETS1 (BIND; HPRD) SRC ETS2 (BIND; HPRD)SRC EVL (CCSB; GRID; HPRD) SRC FARP2 (HPRD) SRC FASLG (GRID; HPRD) SRCFCER1G (reactome) SRC FHIT (HPRD) SRC FOXO1 (GRID; HPRD) SRC FRS2 (HPRD)SRC FYB (HPRD) SRC GAB2 (GRID; HPRD) SRC GAB3 (GRID; HPRD) SRC GFAP(CCSB; GRID; HPRD) SRC GIT1 (GRID; HPRD) SRC GJA1 (BIND; CCSB; GRID;HPRD; reactome) SRC GJB1 (BIND; HPRD) SRC GNB2L1 (BIND; CCSB; GRID;HPRD) SRC GP2 (HPRD) SRC GP6 (reactome) SRC GRB10 (HPRD) SRC GRB2 (CCSB;GRID; HPRD) SRC GRIN2A (GRID; HPRD) SRC GRIN2B (GRID; HPRD) SRC GRLF1(HPRD) SRC GTF2I (HPRD) SRC GUCY2C (HPRD; MINT) SRC HLA-A (HPRD) SRCHLA-B (HPRD) SRC HNF1A (GRID; HPRD) SRC HNRPK (BIND; CCSB; GRID; HPRD)SRC HRAS (HPRD; MINT; reactome) SRC HSP90AA1 (CCSB; DIP; GRID; HPRD) SRCIKBKB (HPRD; IntAct) SRC IKBKG (HPRD; IntAct) SRC IL6R (GRID; HPRD) SRCINPPL1 (HPRD) SRC ITGB3 (HPRD) SRC JUP (HPRD) SRC KCNA5 (HPRD) SRC KCNB1(HPRD) SRC KCNQ5 (HPRD) SRC KHDRBS1 (BIND; GRID; HPRD; IntAct; MDC;MINT) SRC KIFAP3 (CCSB; GRID; HPRD) SRC KRAS (reactome) SRC LCT (MINT)SRC LRP1 (HPRD) SRC MAPK15 (HPRD) SRC MPZL1 (CCSB; GRID; HPRD) SRC MUC1(BIND; GRID; HPRD; MINT) SRC MYLK (GRID; HPRD) SRC NCOA6 (GRID) SRC ND2(GRID; HPRD) SRC NDUFS1 (BIND) SRC NFKBIA (HPRD) SRC NMT1 (HPRD) SRCNOS2A (HPRD) SRC NPHS1 (GRID; HPRD) SRC NRAS (reactome) SRC P2RY2 (HPRD)SRC PAK2 (HPRD) SRC PDE4D (BIND) SRC PDE6G (GRID; HPRD) SRC PDPK1 (HPRD)SRC PECAM1 (GRID; HPRD) SRC PELP1 (GRID; HPRD) SRC PGR (HPRD) SRC PIK3R1(GRID; HPRD) SRC PKD1 (GRID; HPRD) SRC PLCG1 (GRID; HPRD) SRC PLSCR1(GRID) SRC PPARD (HPRD) SRC PRKACA (HPRD) SRC PRKCA (HPRD) SRC PRKCD(HPRD; MINT) SRC PRKCE (GRID; HPRD) SRC PRKCH (HPRD) SRC PRKCI (HPRD)SRC PRKCZ (CCSB; GRID; HPRD) SRC PRKD1 (HPRD) SRC PTPN1 (HPRD) SRCPTPN11 (HPRD) SRC PTPN18 (HPRD) SRC PTPN2 (BIND; HPRD) SRC PTPN21 (GRID;HPRD) SRC PTPN6 (HPRD) SRC PTPRA (HPRD) SRC PTPRE (HPRD) SRC PXN (GRID;HPRD) SRC RAF1 (CCSB; GRID; HPRD) SRC RALA (reactome) SRC RALB(reactome) SRC RALGDS (reactome) SRC RASA1 (CCSB; GRID; HPRD) SRC RGS16(BIND; HPRD) SRC RICS (BIND; GRID; HPRD) SRC SH2D2A (BIND) SRC SH2D3C(HPRD) SRC SH3BP1 (CCSB; GRID; HPRD) SRC SH3PXD2A (GRID; HPRD) SRC SHB(BIND; GRID; HPRD) SRC SHC1 (BIND; CCSB; GRID; HPRD) SRC SKAP1 (GRID;HPRD) SRC SLC9A2 (GRID; HPRD) SRC SMARCB1 (GRID; HPRD) SRC SMARCE1(GRID; HPRD) SRC SNURF (GRID) SRC SPTAN1 (HPRD) SRC SRF (GRID; HPRD) SRCSTAP2 (HPRD) SRC STAT1 (CCSB; GRID: HPRD) SRC STAT3 (CCSB; GRID: HPRD)SRC STAT5A (CCSB; GRID; HPRD) SRC STAT5B (HPRD) SRC STAT6 (HPRD) SRCSUPT4H1 (BIND) SRC SYK (CCSB; GRID; HPRD; reactome) SRC SYN1 (GRID;HPRD) SRC TERT (HPRD) SRC TIAM1 (GRID; HPRD) SRC TLR3 (MINT) SRCTNFRSF11A (HPRD) SRC TNFSF11 (HPRD) SRC TRAF1 (HPRD) SRC TRAF3 (HPRD)SRC TRAF6 (CCSB; GRID; HPRD) SRC TRAT1 (HPRD) SRC TRIP6 (CCSB; GRID;HPRD) SRC TRPC6 (GRID; HPRD) SRC TRPV4 (GRID; HPRD) SRC TUB (GRID; HPRD)SRC TXK (HPRD) SRC TYRO3 (GRID; HPRD) SRC VCL (HPRD) SRC VIL1 (HPRD) SRCWAS (CCSB; GRID; HPRD) SRC WBP11 (HPRD) SRC WT1 (HPRD) SRC YTHDC1 (GRID;HPRD) SRC YWHAB (CCSB; GRID; HPRD) SRC YWHAE (CCSB; GRID; HPRD) SRCYWHAG (CCSB; GRID; HPRD) SRC YWHAH (GRID; HPRD) SRM MAX (BIND) SYK BLNK(CCSB; GRID; HPRD) SYK CBL (BIND; GRID; HPRD; MINT) SYK CBLB (BIND;CCSB; GRID; HPRD) SYK CD19 (GRID; HPRD) SYK CD22 (GRID; HPRD; IntAct)SYK CD3E (GRID; HPRD) SYK CD79A (GRID; HPRD) SYK CD79B (HPRD) SYK COL1A1(reactome) SYK COL1A2 (reactome) SYK CRKL (BIND; GRID; HPRD) SYK CSF2RB(HPRD) SYK CSF3R (GRID; HPRD) SYK CTTN (CCSB; GRID; HPRD) SYK DBNL(HPRD) SYK DTYMK (BIND) SYK DUSP3 (HPRD) SYK EPOR (GRID; HPRD) SYK EZR(BIND; CCSB; GRID) SYK FCER1G (GRID; HPRD; IntAct; reactome) SYK FCGR1A(CCSB; GRID; HPRD) SYK FCGR2A (HPRD) SYK GAB2 (HPRD) SYK GP6 (reactome)SYK GRB2 (CCSB; GRID; HPRD) SYK HCLS1 (HPRD) SYK HMGCS2 (BIND) SYKHNRNPU (GRID; HPRD) SYK IL15RA (HPRD) SYK IL2RB (HPRD) SYK ITGB2 (HPRD;IntAct) SYK JUN (BIND) SYK LAT (CCSB; GRID; HPRD) SYK LAX1 (HPRD) SYKLCP2 (CCSB; GRID; HPRD) SYK MAP4K1 (HPRD) SYK MAPK3 (HPRD) SYK MAX(BIND) SYK MS4A2 (GRID; HPRD) SYK MSN (BIND) SYK MYC (BIND) SYK NFAM1(HPRD) SYK NFKBIB (BIND) SYK PAG1 (GRID; HPRD) SYK PIK3AP1 (HPRD) SYKPIK3R1 (HPRD; MINT) SYK PIK3R2 (HPRD; MINT) SYK PLCG1 (DIP; GRID; HPRD)SYK PLCG2 (HPRD; reactome) SYK POU2AF1 (IntAct) SYK PRKCA (HPRD) SYKPRKD1 (HPRD) SYK PTPN6 (CCSB; GRID; HPRD) SYK PXN (GRID; HPRD) SYK RASA1(HPRD) SYK RPS6KA1 (HPRD) SYK RPS6KA2 (DIP) SYK SELPLG (HPRD) SYK SH2B2(HPRD) SYK SH3BP2 (GRID; HPRD) SYK SHC1 (HPRD) SYK SIT1 (GRID; HPRD) SYKSLA (CCSB; GRID; HPRD) SYK SLC4A1 (HPRD) SYK SNCA (HPRD) SYK STAT1(BIND; HPRD) SYK STAT3 (CCSB; GRID; HPRD) SYK STAT5A (CCSB; GRID; HPRD)SYK TAF1 (BIND) SYK TLR4 (HPRD) SYK TRAF6 (CCSB; GRID; HPRD) SYK TUBA1A(CCSB; GRID; HPRD) SYK TUBA4A (HPRD) SYK TYROBP (HPRD) SYK UBB (BIND;CCSB; GRID; HPRD) SYK UCP2 (BIND) SYK VAV1 (CCSB; GRID; HPRD) SYK VAV2(HPRD) TEC DOK1 (HPRD) TEC EPOR (HPRD) TEC GNA12 (GRID; HPRD) TEC IL3RA(HPRD) TEC PIK3R1 (HPRD) TEC PIK3R3 (GRID; HPRD) TEC PIP4K2A (HPRD) TECPIP5K1A (HPRD) TEC PLCG1 (HPRD) TEC PLCG2 (HPRD) TEC PLK4 (GRID; HPRD)TEC PRLR (GRID; HPRD) TEC PTPN18 (HPRD) TEC PTPN21 (GRID; HPRD) TEC SHC1(HPRD) TEC SOCS1 (GRID; HPRD) TEC STAP1 (HPRD) TEC VAV1 (GRID; HPRD) TECWAS (GRID; HPRD) TEK ANGPT1 (DIP; GRID; HPRD) TEK ANGPT2 (BIND; DIP;GRID; HPRD) TEK ANGPT4 (BIND; GRID; HPRD) TEK ANGPTL1 (BIND; GRID; HPRD)TEK DOK2 (GRID; HPRD) TEK DOK4 (HPRD) TEK GRB14 (GRID; HPRD) TEK GRB2(GRID; HPRD) TEK GRB7 (GRID; HPRD) TEK PIK3R1 (GRID; HPRD) TEK PTPN11(GRID; HPRD) TEK PTPRB (HPRD) TEK SHC1 (HPRD) TEK SOCS1 (BIND; GRID;HPRD) TEK STAT5A (HPRD) TEK STAT5B (HPRD) TEK TIE1 (GRID; HPRD) TEKTNIP2 (HPRD) TIE1 PIK3R1 (GRID; HPRD) TIE1 PTPN11 (CCSB; GRID; HPRD)TNK1 PLCG1 (GRID; HPRD) TNK1 SFN (GRID) TNK2 ARSE (HPRD; MDC) TNK2 CDC42(CCSB; GRID; HPRD) TNK2 CLTC (GRID; HPRD) TNK2 CSPG4 (GRID; HPRD) TNK2GRB2 (CCSB; GRID; HPRD) TNK2 HSH2D (GRID; HPRD) TNK2 HSP90AB2P (HPRD)TNK2 ITFG2 (HPRD; MDC) TNK2 MCF2 (GRID; HPRD) TNK2 MERTK (HPRD) TNK2NCK1 (CCSB; GRID; HPRD) TNK2 RASGEF1C (HPRD; MDC) TNK2 RASGRF1 (GRID;HPRD) TNK2 RPL18A (HPRD; MDC) TNK2 SEZ6 (HPRD; MDC) TNK2 SFRS5 (HPRD;MDC) TNK2 SNX9 (HPRD) TNK2 VTI1B (HPRD; MDC) TNK2 WWOX (HPRD) TXK CCR5(GRID; HPRD) TXK CTLA4 (HPRD) TXK LCP2 (HPRD) TXK SH2D2A (HPRD) TXK TH1L(HPRD) TXK YTHDC1 (GRID; HPRD) TYK2 CBL (BIND; GRID; HPRD) TYK2 CRKL(GRID; HPRD) TYK2 GHR (GRID; HPRD) TYK2 GNB2L1 (BIND; CCSB; GRID; HPRD;MINT) TYK2 IFNAR1 (BIND; CCSB; DIP; GRID; HPRD; MINT) TYK2 IFNAR2 (BIND;HPRD) TYK2 IL12RB1 (BIND) TYK2 IL13RA1 (CCSB; GRID; HPRD) TYK2 IL6ST(DIP; GRID; HPRD) TYK2 IRS1 (GRID; HPRD) TYK2 IRS2 (GRID; HPRD) TYK2JAKMIP1 (BIND; HPRD) TYK2 MAX (BIND) TYK2 MYC (BIND) TYK2 PIK3R1 (GRID;HPRD) TYK2 PLAUR (BIND; DIP; HPRD) TYK2 PRMT5 (GRID; HPRD) TYK2 PTAFR(HPRD) TYK2 PTPN1 (CCSB; GRID; HPRD) TYK2 PTPN6 (CCSB; GRID; HPRD) TYK2PTPRC (HPRD) TYK2 STAM2 (CCSB; GRID; HPRD) TYK2 STAT1 (GRID; HPRD; MINT)TYK2 STAT2 (HPRD) TYK2 TAF1 (BIND) TYK2 VAV1 (CCSB; GRID; HPRD) TYK2XRCC5 (GRID; HPRD) TYRO3 GAS6 (GRID; HPRD) TYRO3 PIK3R1 (GRID; HPRD)TYRO3 PROS1 (GRID; HPRD) TYRO3 YES1 (GRID; HPRD) YES1 ADAM12 (GRID;HPRD) YES1 ADAM15 (MINT) YES1 CD2AP (GRID; HPRD; MINT) YES1 CD36 (GRID;HPRD) YES1 CD46 (GRID; HPRD) YES1 CDCP1 (BIND) YES1 CKAP4 (IntAct) YES1DLG4 (GRID; HPRD) YES1 DOK1 (HPRD) YES1 GP2 (HPRD) YES1 GP6 (HPRD) YES1ITGB4 (HPRD) YES1 NPHS1 (GRID; HPRD) YES1 OCLN (GRID; HPRD) YES1 PECAM1(GRID; HPRD) YES1 PTPRE (HPRD) YES1 RASA1 (GRID; HPRD) YES1 RPL10 (GRID;HPRD) YES1 TAF1 (BIND) YES1 TP53BP2 (GRID; HPRD) YES1 TRPV4 (GRID; HPRD)YES1 YAP1 (GRID) YES1 ZNF512B (BIND; MINT) ZAP70 ACP1 (CCSB; GRID; HPRD)ZAP70 CARD11 (HPRD) ZAP70 CBL (GRID; HPRD) ZAP70 CBLB (CCSB; GRID; HPRD)ZAP70 CD247 (BIND; CCSB; GRID; HPRD) ZAP70 CD3E (GRID; HPRD) ZAP70 CD5(GRID; HPRD) ZAP70 CD79B (HPRD) ZAP70 CRK (BIND; CCSB; GRID; HPRD) ZAP70CRKL (BIND) ZAP70 DBNL (CCSB; GRID; HPRD) ZAP70 DEF6 (HPRD) ZAP70 DUSP3(HPRD) ZAP70 FCGR3A (CCSB; GRID; HPRD) ZAP70 FCRL3 (GRID; HPRD) ZAP70GAB2 (GRID; HPRD) ZAP70 GRB2 (HPRD) ZAP70 HSP90AA1 (MINT) ZAP70 IFNAR1(HPRD) ZAP70 LAT (HPRD; MINT) ZAP70 LAX1 (HPRD) ZAP70 LCP2 (HPRD) ZAP70MUC1 (HPRD) ZAP70 NFAM1 (HPRD) ZAP70 PAG1 (GRID; HPRD) ZAP70 PLCG1(GRID; HPRD) ZAP70 PRLR (GRID; HPRD) ZAP70 PTPN3 (HPRD) ZAP70 PTPN6(CCSB; GRID; HPRD) ZAP70 PTPRC (GRID; HPRD) ZAP70 RASA1 (CCSB; GRID;HPRD) ZAP70 SH2B3 (HPRD) ZAP70 SH3BP2 (GRID; HPRD) ZAP70 SHB (GRID;HPRD) ZAP70 SHC1 (BIND; CCSB; GRID; HPRD) ZAP70 SIT1 (GRID; HPRD) ZAP70SLA (CCSB; GRID; HPRD) ZAP70 SLA2 (GRID; HPRD) ZAP70 SLAMF6 (GRID; HPRD)ZAP70 SOS1 (GRID; HPRD) ZAP70 TUBA4A (CCSB; GRID; HPRD) ZAP70 TUBB(HPRD) ZAP70 TUBB2A (HPRD) ZAP70 TYROBP (HPRD) ZAP70 UBE2L3 (HPRD) ZAP70VAV1 (CCSB; GRID; HPRD) ZAP70 WIPF1 (HPRD)

A list of the interacting proteins of Table 5 followed by theiraccession numbers (in parenthesis) are as follows:

AATK (205986_at), ABI1 (209027_s_at 209028_s_at), ABI2 (225098_at209856_x_at 207268_x_at 225112_at 211793_s_at), ABL1 (202123_s_at), ABL2(231907_at 206411_s_at 226893_at), ACP1 (1554808_at 201630_s_at215227_x_at 201629_s_at), ACPP (237030_at 231711_at 204393_s_at), ACTA1(203872_at), ACTB (AFFX-HSAC07X00351_(—)3_at 213867_x_atAFFX-HSAC07X00351_M_at AFFX-HSAC07/X00351_(—)5_at 224594_x_at200801_x_at), ACTN1 (208637_x_at 208636_at), ADAM10 (202604_x_at233538_s_at 214895_s_at), ADAM12 (202952_s_at 213790_at 204943_at),ADAM15 (217007_s_at 1555896_a_at), ADAM17 (205746_s_at 205745_x_at),ADCYAP1 (230237_at 206281_at), ADCYAP1R1 (207151_at 236373_at 226690_at242547_at), ADD2 (205268_s_at 237336_at 206807_s_at), ADH6 (214261_s_at207544_s_at), ADORA2A (205013_s_at), ADRB2 (206170_at), ADRB3 (206812_at217303_s_at), ADRBK1 (201401_s_at), AFAP1 (203563_at), AFAP1L2(226829_at), AGK (1555610_at 222132_s_at 218568_at), AGTR1 (205357_s_at208016_s_at), AGTR2 (207293_s_at 222321_at 207294_at), AHSG (210929_s_at204551_s_at), AKAP5 (230846_at 207800_at), AKT1 (207163_s_at), ALCAM(1569362_at 240655_at 201951_at 201952_at), ALK (208211_s_at208212_s_at), AMH (206516_at), ANGPT1 (241119_at 1552939_at 205609_at205608_s_at), ANGPT2 (237261_at 205572_at 236034_at 211148_s_at), ANGPT4(221134_at), ANGPTL1 (239183_at 231773_at 224339_s_at), ANKRD11(238538_at 234701_at 227661_at 219437_s_at 228356_at 231999_at), ANKS1A(212747_at), ANXA1 (201012_at), ANXA2 (210427_x_at 208816_x_at213503_x_at 201590_x_at 1568126_at), ANXA5 (200782_at), ANXA6(200982_s_at), AP2A1 (234068_s_at 223237_x_at 229617_x_at), AP2A2(212159_x_at 211779_x_at 215764_x_at 212161_at), AP2B1 (200615_s_at200612_s_at), AP2M1 (200613_at), AP2S1 (202120_x_at 211047_x_at208074_s_at), APBA3 (205146_x_at 215148_s_at), APBB1 (202652_at), APBB2(213419_at 212972_x_at 212985_at 216747_at 216750_at 40148_at212970_at), APBB3 (204650_s_at), APC (215310_at 203526_s_at 203525_s_at203527_s_at 216933_x_at), APP (200602_at 211277_x_at 237571_at214953_s_at 243314_at), APPL1 (222538_s_at 218158_s_at), AQP1 (209047_at207542_s_at), AR (211621_at 211110_s_at), ARAF (201895_at), AREG(205239_at), ARF1 (208750_s_at 200065_s_at 244504_x_at), ARF4(201096_s_at 201097_s_at), ARHGAP1 (216689_x_at 241419_at 217153_at202117_at), ARHGAP21 (241701_at 224764_at), ARHGAP26 (226576_at205069_s_at 205068_s_at 215955_x_at), ARHGAP5 (233849_s_at 217936_at235635_at 1552627_a_at), ARHGEF12 (237398_at 234541_s_at 233620_at233621_s_at 201335_s_at 234129_at 201333_s_at 201334_s_at). ARHGEF15(217348_x_at 205507_at), ARHGEF6 (209539_at), ARID3A (205865_at), ARR3(207136_at), ARRB1 (228444_at 222912_at 49111_at 222756_s_at 43511_s_at218832_x_at 221861_at), ARSE (205894_at), ATF2 (1555146_at 205446_s_at),ATF3 (1554420_at 1554980_a_at 202672_s_at), ATF71P (231825_x_at218987_at 216197_at 216198_at), ATG12 (213930_at 213026_at 204833_at),ATIC (208758_at), ATM (1554631_at 210858_x_at 208442_s_at 212672_at1570352_at 1553387_at), ATP1A1 (220948_s_at), ATP1B1 (227556_at201243_s_at 201242_s_at), ATP2B4 (212136_at 205410_s_at 212135_s_at),ATP5C1 (205711_x_at 208870_x_at 213366_x_at), ATP5H (210149_s_at1555998_at), ATP50 (1564482_at 200818_at), ATP6V1H (1557585_at1557586_s_at 221504_s_at), ATR (209902_at 233288_at 209903_s_at), ATXN1(236404_at 1559249_at 203232_s_at 203231_s_at 242230_at 230507_at236802_at), AXL (202685_s_at 202686_s_at), BAG1 (202387_at 229720_at211475_s_at), BANK1 (1558662_s_at 222915_s_at 219667_s_at), BCAR1(223116_at), BCAS2 (203053_at), BCL2 (203685_at 207005_s_at 207004_at203684_s_at 237837_at), BCL2L1 (215037_s_at 212312_at 206665_s_at), BCL3(204908_s_at 204907_s_at), BCR (226602_s_at 202315_s_at 217223_s_at),BDNF (239367_at 244503_at 206382_s_at), BIN1 (214643_x_at 202931_at214439_at 210202_s_at 210201_at), XIAP (225858_s_at 235222_x_at206536_s_at 225859_at 243026_at 228363_at), BLK (244394_at 236820_at206255_at 210934_at), BLNK (244172_at 207655_s_at 243867_at), BMPR2(238516_at 225144_at 210214_s_at 209920_at 231873_at), BMX (206464_at242967_at), BNIP2 (209308_s_at), BRAF (206044_s_at 243829_at), BRCA1(211851_at 204531_s_at), BTC (241412_at 207326_at), BTK (205504_at),C13orf34 (219544_at), C14orf4 (not_found), BEND5 (219670_at), C2orf44(219120_at), C3 (217767_at), C6orf47 (204968_at), CABLES1 (225531_at225532_at), CABLES2 (226004_at), CACNA1D (207998_s_at 210108_at), CALM1(213710_s_at 213688_at 211985_s_at 211984_at 209563_x_at 200653_s_at200655_s_at), CALM2 (207243_s_at). CALM5 (1563431_at 200622_at200623_s_at), CAMK2A (213108_at 207613_s_at), CAMK2G (212669_at212757_s_at), CAMLG (203538_at), CARD11 (223514_at), CASP1 (211366_x_at209970_x_at 211368_s_at 211367_s_at 1552703_s_at), CASP3 (202763_at),CASP7 (207181_s_at), CASP8 (207686_s_at 1553306_at 213373_s_at), CASP9(237451_x_at 240437_at 203984_s_at 210775_x_at), CAT (201432_at211922_s_at), CAV1 (212097_at 203065_s_at), CAV2 (213426_s_at 203323_at203324_s_at), CAV3 (208204_s_at), CBL (225231_at 229010_at 206607_at243475_at 225234_at), CBLB (208348_s_at 209682_at 227900_at), CBLC(220638_s_at 223668_at), CCDC17 (236320_at), CCNA2 (213226_at203418_at), CCND1 (208712_at 208711_s_at), CCR1 (205099_s_at 205098_at),CCR2 (207794_at 206978_at), CCR3 (208304_at), CCR5 (206991_s_at), CD19(206398_s_at), CD2 (205831_at), CD22 (220674_at 204581_at 38521_at217422_s_at), CD226 (207315_at), CD24 (216379_x_at 208651_at 209771_x_at266_s_at 209772_s_at 208650_s_at), CD247 (210031_at), CD28 (206545_at211856_x_at 211861_at), CD2AP (203593_at 236257_at), CD33 (206120_at),CD36 (228766_at 209554_at 206488_s_at 209555_s_at), CD38 (234187_at205692_s_at), CD3E (205456_at), CD4 (216424_at 203547_at), CD40(205153_s_at 35150_at 222292_at 215346_at), CD44 (1565868_at 212014_x_at229221_at 212063_at 209835_x_at 217523_at 1557905_s_at 210916_s_at204490_s_at 216056_at 204489_s_at), CD46 (208783_s_at 211574_s_at207549_at), CD47 (213856_at 213055_at 213857_s_at 226016_at 211075_s_at242974_at 227259_at), CD48 (237759_at 204118_at), CD5 (230489_at206485_at), CD55 (1555950_a_at 201925_s_at 201926_s_at), CD59 (228748_at200983_at 212463_at 200984_s_at 200985_s_at), CD63 (200663_at), CD79A(205049_s_at 1555779_a_at), CD79B (205297_s_at 1555748_x_at 1555746_at),CD81 (200675_at), CD82 (228910_at 203904_x_at), CD8A (205758_at), CD8B(230037_at 215332_s_at 207979_s_at), CD9 (201005_at), CDAN3 (not_found),CDK1 (231534_at 203214_at 210559_s_at 203213_at), CDC25A (1555772_a_at204696_s_at 204695_at), CDC25C (217010_s_at 205167_s_at), CDC37(209953_s_at), CDC42 (210232_at 214230_at 208728_s_at 208727_s_at),CDCP1 (218451_at 1554110_at 234932_s_at), CDH1 (201131_s_at201130_s_at), CDH2 (203440_at 203441_s_at), CDH5 (204677_at), CDK2(211804_s_at 204252_at 211803_at), CDK4 (202246_s_at), CDK5(204247_s_at), CDK5R1 (204996_s_at 204995_at), CDKN1B (209112_at),CEACAM1 (211889_at 209498_at 206576_s_at 210610_at 211883_at), CEACAM3(208052_x_at 210789_x_at), CEBPB (212501_at), CENPF (207828_s_at209172_s_at), ARAP1 (34206_at 212516_at), CHGB (204260_at), CHN1(212624_s_at), CHN2 (207486_x_at 213385_at 211419_s_at), CHUK(209666_s_at), CIB1 (201953_at). CISH (221223_at 223377_x_at223961_s_at), CKAP4 (200998_s_at 200999_s_at 226526_s_at), CLDN4(201428_at), CLTA (1560434_x_at 200960_x_at 1560433_at 216295_s_at204050_s_at), CLTC (200614_at), CLTCL1 (205944_s_at), CMTM3 (1555704_at1555705_a_at 224733_at), CNN1 (203951_at), CNN3 (201445_at), CNR1(213436_at 208243_s_at 207940_x_at), CNTN1 (241190_at 211203_s_at227202_at), CNTNAP1 (219400_at), COL11A1 (229271_x_at 37892_at204320_at), COL18A1 (209082_s_at 209081_s_at), COL1A1 (1556499_s_at202310_s_at 202312_s_at 217430_at 202311_s_at), COL1A2 (202404_s_at202403_s_at), COL2A1 (217404_s_at 213492_at), COL3A1 (211161_s_at215076_s_at 201852x_at), COL5A2 (221729_at 221730_at), COLQ (206073_at),COPA (214336_s_at 1559862_at 208684_at), COPB1 (201359_at 201358_s_at),PTGS2 (204748_at 1554997_a_at), COX6C (201754_at), COX7B (202110_at),CPSF4 (206688_s_at), CREB1 (204313_s_at 225565_at 214513_s_at204314_s_at 237289_at 243625_at 204312_at 225572_at), CREBBP(211808_s_at 202160_at 228177_at 237239_at), CRK (202224_at202226_s_at), CRKL (206184_at 212180_at), CRLF2 (208303_s_at), CSF1(211839_s_at 210557_at 207082_at 209716_at), CSF1R (203104_at), CSF2RA(210340_s_at 211287_at 211286_at 207085_at), CSF2RB (205159_at). CSF3R(203591_s_at 1553297_a_at), CSK (202329_at), CSNK2B (201390_s_at), CSPG4(214297_at 204736_s_at), CTGF (209101_at), CTLA4 (221331_at 231794_at234895_at 236341_at 234362_s_at), CTNNB1 (1554411_at 201533_at), CTNND1(211240_x_at 208862_s_at 208407_s_at 1557944_s_at), CTNND2 (209617_s_at209618_at), CTR9 (202060_at), CTSK (202450_s_at), CTTN (201059_at214782_at 214074_s_at), CXCR4 (211919_s_at 217028_at 209201_x_at),CYP2D6 (207498_s_at 217468_at 215809_at), DAB1 (228329_at 220611_at),DAB2 (210757_x_at 201278_at 232898_at 201279_s_at 201280_s_at), DAG1(212128_s_at 205417_s_at), DAPK3 (203891_s_at 228681_x_at 203890_s_at),DAPP1 (222859_s_at 222858_s_at 219290_at), DBNL (222429_at), DCC(238914_at 206939_at), DCN (239786_at 201893_x_at 211896_s_at211813_x_at 209335_at 229554_at), DDB1 (208619_at), DDB2 (203409_at),ASAP1 (231205_at 237082_at 224790_at 224791_at 236533_at 221039_s_at224796_at), ASAP2 (206414_s_at). DDR1 (208779_at 207169_x_at 210749_at1007_s_at), DDB2 (227561_at 205168_at), DEF6 (221293_s_at 226659_at),DEGS1 (207431_s_at 209250_at), DGKA (211272_s_at 203385_at), DGKZ(207556_s_at 239342_at), DIABLO (219350_s_at), DLG1 (229703_at 202515_at215988_s_at 217208_s_at 202516_s_at 202514_at), DLG2 (228973_at206253_at), DLG3 (241839_at 207732_s_at 212729_at 212727_at 212728_at),DLG4 (204592_at 210684_s_at), DLGAP3 (231151_at), DNAJA3 (205963_s_at1554078_s_at), DNM1 (215116_s_at 217341_at), DNM2 (202253_s_at216024_at), DOK1 (216835_s_at 211121_s_at), DOK2 (214054_at), DOK3(223553_s_at 220320_at), DOK4 (207747_s_at 209690_s_at 209691_s_at),DOK5 (214844_s_at 1554863_s_at), DOK6 (236290_at 242867_at 241729_at231980_at), DOK7 (240633_at), DPM2 (209391_at), DPYSL5 (224100_s_at222797_at), DRD4 (208215_at), DTYMK (1553984_s_at 203270_at), DUSP3(201537_s_at 201536_at 201538_s_at), DYNLL1 (200703_at), E2F4(38707_r_at 202248_at), S1PR1 (239401_at 204642_at 244422_at), EFNA1(202023_at), EFNA2 (208256_at 1553573_s_at), EFNA3 (210132_at), EFNA4(205107_s_at), EFNA5 (227955_s_at 214036_at 207301_at 233814_at), EFNB1(202711_at), EFNB2 (202669_s_at 202668_at), EFNB3 (210883_at 205031_at),EFS (210880_s_at 204400_at), EGF (206254_at), EGFR (211550_at 210984_at211551_at 211607_x_at 201984_s_at 201983_s_at 1565483_at 1565484_at),EGR1 (201694_s_at 201693_s_at 227404_s_at), EHD1 (222221_x_at209039_x_at 209038_s_at 209037_s_at 208112_x_at), EIF2AK2 (204211_x_at),EIF3E (236989_at 208697_s_at 235429_at), EIF4B (211938_at 219599_at211937_at), ELF3 (229842_at 201510_at 210827_s_at), ELMO1 (204513_s_at),ELP2 (231713_s_at 232503_at), ENPP1 (228952_at 229088_at 205066_s_at205065_at), EP300 (213579_s_at 202221_s_at), EPB41 (207793_s_at214530_x_at 225051_at 1554481_a_at), EPHA1 (215804_at 205977_s_at),EPHA2 (203499_at), EPHA3 (206071_s_at 206070_s_at 211164_at), EPHA4(227449_at 228948_at 229374_at 206114_at), EPHA5 (215664_s_at 216837_at237939_at), EPHA6 (233184_at 1561396_at), EPHA7 (1554629_at 229288_at238533_at 206852_at), EPHA8 (231796_at 1554069_at), EPHB1 (230425_at211898_s_at 210753_s_at), EPHB2 (209588_at 209589_s_at 211165_at210651_s_at), EPHB3 (204600_at 1438_at), EPHB4 (202894_at 216680_s_at),EPHB6 (204718_at), EPOR (209962_at 216999_at 209963_s_at 396_f_at37986_at 215054_at), EPPK1 (232164_s_at 208156_at 232165_at), EPS15(217886_at 234277_at 217887_s_at 234278_at), EPS8 (202609_at), ERBB2(210930_s_at 216836_s_at), ERBB21P (217941_s_at 222473_s_at), ERBB3(1563252_at 202454_s_at 226213_at 1563253_s_at), ERBB4 (206794_at241581_at 233494_at 214053_at 233498_at), EREG (1569583_at 205767_at),ERF (203643_at), ERRFI1 (224657_at), ESR1 (217163_at 211627_x_at205225_at 215552_s_at 211233_at 211235_s_at 211234_at), ESR2(211120_x_at 211119_at 240777_at 211117_at 211118_at 210780_at), ETS1(224833_at 1555355_a_at 241435_at 214447_at), ETS2 (242784_at201329_s_at 201328_at), EVL (244375_at 217838_s_at 227232_at), EWSR1(217622_at 210011_s_at 210012_s_at 229966_at 209214_s_at), EZR(217234_s_at 208622_s_at 208621_s_at 217230_at 208623_s_at), FABP4(203980_at), FARP2 (1558855_at 1554337_at 204511_at), FAS (216252_x_at215719_at 204780_s_at 204781_s_at), FASLG (211333_s_at 210865_at), FAU(200019_s_at), FBP1 (209696_at), FCAR (211306_s_at 211816_x_at211305_x_at 207674_at 211307_s_at), FCER1G (204232_at 1554899_s_at),FCER2 (206759_at 206760_s_at), FCGR1A (214511_at 216950_s_at 216951_at),FCGR2A (211395_x_at 210992_x_at 203561_at 1565674_at 1565673_at), FCGR2B(211395_at 210889_s_at), FCGR3A (204007_at 204006_s_at), FCRL3(231093_at 1553196_a_at), FDPS (201275_at), FER (227579_at 206412_at),FES (205418_at), FGA (205650_s_at 205649_s_at), FGF1 (205117_at208240_s_at 1552721_a_at), FGF10 (231762_at), FGF16 (221374_at), FGF17(221376_at), FGF18 (231382_at 211485_s_at 206987_at 211029_at), FGF19(223761_at), FGF2 (204422_s_at 204421_s_at), FGF20 (220394_at), FGF22(221315_s_at), FGF23 (221166_at), FGF3 (214571_at), FGF4 (1552982_a_at),FGF5 (210310_s_at 210311_at 208378_at), FGF6 (208417_at), FGF7(1555103_s_at 205782_at 1555102_at 1554741_s_at), FGF8 (208449_s_at),FGF9 (239178_at 206404_at), FGFR1 (215404_x_at 207822_at 226705_at211535_s_at 210973_s_at 207937_x_at), FGFR2 (211399_at 203639_s_at208225_at 208234_at 211398_at 211401_s_at 208228_s_at 203638_s_at211400_at), FGFR3 (204380_s_at 204379_s_at), FGFR4 (1554961_at211237_s_at 204579_at 1554962_a_at), FGR (208438_s_at), FHIT(206492_at), FIGF (206742_at), FIZ1 (228663_at 226967_at), FLOT1(210142_at 208749_at 208748_s_at), FLOT2 (201350_at 211299_s_at), FLT1(210287_s_at 226497_s_at 222033_s_at 226498_at), FLT3 (206674_at),FLT3LG (206980_s_at 210607_at), FLT4 (234379_at 210316_at 229902_at),FLYWCH1 (223950_s_at 234106_s_at 225952_at), FN1 (211719_at 235629_at214702_at 214701_s_at 212464_s_at 1558199_at 216442_at 210495_at), FNBP4(235101_at 212232_at), FOLR1 (204437_s_at), FOS (209189_at), FOXO1(228484_s_at 239728_at 202724_s_at 202723_s_at), FRK (207178_s_at), FRS2(226045_at 221308_at 238486_at), FRS3 (219907_at), FYB (224148_at211795_s_at 205285_s_at 211794_at 227266_s_at), FYN (217697_at210105_s_at 216033_s_at), FZD2 (210220_at 238129_s_at), FZD5(221245_s_at 206136_at), FZD8 (216587_s_at 224325_at 227405_s_at), GAB1(225998_at 214987_at 229114_at 1560382_at 226002_at 207112_s_at), GAB2(203853_s_at), GAB3 (241259_at 228410_at), CHST15 (244874_at 203066_at),GAPDH (AFFX-HUMGAPDH/M33197_(—)3_at AFFX-HUMGAPDHM33197_(—)5_atAFFX-HUMGAPDH/33197_M_at 212581_x_at 217398_at 213453_x_at), GAS6(202177_at 1598_g_at), GDNF (221359_at 230090_at), GFAP (203539_s_at229259_at 203540_at), GFRA1 (205696_s_at 227550_at 230163_at), GFRA4(234868_s_at 221199_at), GHR (241584_at 205498_at), GIGYF1 (228755_at226768_at 236447_at), GIPC1 (207525_s_at), GIT1 (218030_at), GJA1(201667_at), GJB1 (204973_at), GJC1 (228563_at 208460_at 228776_at243502_at), GLMN (207153_s_at), GLRX (206662_at 209276_s_at), GNA11(214679_x_at 40562_at 204248_at 564_at 213944_at 213766_x_at 221955_at),GNAI2 (221737_at 224681_at), GNA13 (224761_at 227539_at 206917_at),GNAI1 (209576_at 227692_at), GNAI2 (201040_at), GNAQ (211426_at), GNB2L1(200651_at), GNG2 (223943_s_at 224965_at 224964_s_at 1555766_a_at), GP2(208473_s_at 214324_at 206681_at), GP6 (220336_s_at), GPSM3 (204265_s_at214847_s_at), GPT (206709_x_at), GPX1 (200736_s_at), GRAP (229726_at206620_at), GRAP2 (208406_s_at), GRB10 (209410_s_at 215248_at 209409_at210999_s_at), GRB14 (206204_at), GRB2 (223049_at 215075_s_at), GRB7(210761_s_at), GRIA3 (1569290_s_at 208032_s_at 206730_at 230144_at),GRIN1 (205914_s_at 205915_at 211125_x_at 210781_at 210782_x_at), GRIN2A(231384_at 206534_at 242286_at), GRIN2B (237933_at 210411_s_at210412_at), GRIN2D (229883_at 207036_at), GRLF1 (202044_at 229394_s_at202045_s_at 239456_at 202046_s_at), GSK3B (242336_at 209945_s_at226191_at), GSN (214040_s_at 200696_s_at 234240_at 227957_at 234431_at227958_s_at), GSTO1 (1557915_s_at 201470_at), GTF2I (210891_s_at239649_at 229896_at 210892_s_at 201065_s_at), GTF3C1 (202320_at35671_at), GUCY2C (206312_at), GYS1 (201673_s_at), GZMB (210164_at),H3F3A (200080_s_at 213828_at 208755_x_at 213826_s_at 211940_at), HBEGF(38037_at 203821_at 222076_at), HBZ (206647_at), HCK (208018_s_at),HCLS1 (202957_at), HDHD2 (223155_at), HES1 (203395_s_at 203394_s_at237115_at), HESS (239230_at), HGF (210997_at 209960_at 210998_s_at210755_at 209961_s_at), HGFAC (207027_at), HGS (232627_at 210428_s_at),HIST2H4A (207046_at 230795_at), HIST3H3 (208572_at), HIST1H4I(214634_at), HLA-A (221875_x_at 217436_x_at 215313_x_at 211911_x_at204806_x_at 208729_x_at), HLA-B (216526_x_at 211911_at 214459_x_at209140_x_at 208729_at), HMGA1 (206074_s_at 210457_x_at), HMGCS2(204607_at 240110_at), HNF1A (210515_at), HNF4A (214832_at 230914_at208429_x_at 216889_s_at), HNRNPC (214737_x_at 200751_s_at 200014_s_at212626_at 227110_at), HNRNPL (35201_at 202072_at), HNRNPU (225805_at200594_at 200593_s_at 235603_at 216855_s_at), HNRNPA3P1 (211931_s_at206809_s_at 211933_s_at 206808_at), HNRNPK (200097_s_at 200775_s_at),HOXC10 (218959_at), HRAS (212983_at), HSH2D (1552623_at), HSP90AA1(214328_s_at 211969_at 210211_s_at 211968_s_at), HSP90AB2P (1557910_at214359_s_at), HSP90B1 (216449_at 200598_s_at 200599_s_at), HSPA8(210338_s_at 221891_at 224187_x_at 208687_at), HSPD1 (200806_s_at200807_s_at), HTR2A (211616_s_at 244130_at 207135_at), HTR6(1552857_a_at), HTT (202389_s_at 202390_s_at), HYAL2 (206855_s_at), IBTK(210970_s_at), ICAM1 (202637_s_at 215485_s_at 202638_s_at), IFNAR1(225661_at 225669_at 204191_at), IFNAR2 (204785_x_at 230735_at204786_s_at), IFNGR1 (202727_s_at 242903_at 211676_s_at), IFNGR2(201642_at), IGF1 (211577_s_at 209542_at 209541_at 209540_at), IGF1R(208441_at 243358_at 203627_at 225330_at 238544_at 237377_at 237881_at203628_at), IGF2 (210881_s_at 202410_x_at 202409_at), IGFBP3(210095_s_at 212143_s_at), IGHM (212827_at), IKBKB (209342_s_at209341_s_at 211027_s_at), IKBKG (209929_s_at 36004_at), IL10RA(204912_at), IL12RB1 (206890_at 239522_at 1552584_at), IL12RB2(206999_at), IL13RA1 (210904_s_at 201888_s_at 211612_s_at 201887_at),IL15RA (207375_s_at), IL17RD (227997_at 229263_at), IL21R (237753_at221658_s_at 219971_at), IL23R (1561853_a_at 1552912_a_at), IL2RB(205291_at), IL2RG (204116_at), IL3RA (206148_at), IL4R (203233_at),IL5RA (211516_at 211517_s_at 207902_at 210744_s_at), IL6R (205945_at217489_s_at), IL6ST (211000_s_at 212196_at 234474_x_at 214077_x_at204863_s_at 212195_at 234967_at 204864_s_at), IL7R (226218_at205798_at), IL9R (217212_s_at 208164_s_at), IMPDH2 (201892_s_at), INPP5D(1568943_at 233545_at 203331_s_at 203332_s_at), INPPL1 (201598_s_at),INS (206598_at), INSR (227432_s_at 226450_at 213792_s_at 243002_at226212_s_at 226216_at 207851_s_at), INSRR (215776_at), IQGAP1(213446_s_at 210840_s_at 200791_s_at), IRAK1 (201587_s_at 1555784_s_at),IRS1 (238933_at 242979_at 239463_at 235392_at 204686_at), IRS2(236338_at 209185_s_at 209184_s_at), IRS4 (207403_at), ITFG2 (226295_at220589_s_at), ITGA5 (201389_at), ITGAV (202351_at), ITGB1 (1553530 aat211945_s_at 1553678_a_at 215879_at 216190_x_at 216178_at), ITGB2(1555349_a_at 202803_s_at), ITGB3 (204625_s_at 204627_s_at 216261_at211579_at 215240_at 204628_s_at 204626_s_at), ITGB4 (204989_s_at214292_at 230704_s_at 211905_s_at 204990_s_at), ITGB5 (201125_s_at214021_at), ITGB6 (208084_at 208083_s_at), ITK (211339_s_at), ITPR1(1562373_at 203710_at 231329_at 216944_s_at 244090_at 211323_s_at),ITSN1 (209297_at 209298_s_at 210713_at 35776_at 207322_at), JAK1(1552611_a_at 239695_at 1552610_a_at 201648_at), JAK2 (205842_s_at205841_at 1562031_at), JAK3 (211109_at 211108_s_at 207187_at 227677_at),JAKMIP1 (238600_at), JUN (201466_s_at 201464_at 213281_at 201465_s_at),JUP (201015_s_at), KALRN (232717_at 206078_at 227750_at 236651_at205635_at), KCNA2 (239118_at 208564_at), KCNA5 (206762_at), KCNB1(211006_s_at), KCNMA1 (221584_s_at 228414_at 221583_s_at 214921_at),KCNQ5 (244623_at 223891_at), KDR (203934_at), KHDRBS1 (200040_at201488_at), KIAA1377 (232166_at 236325_at 235956_at), KIDINS220(1557246_at 214932_at 212163_at 212162_at), KIFAP3 (203333_at), KIR2DL3(211410_x_at 211397_x_at 216907_x_at 211532_x_at 207314_x_at 208122_x_at208203_x_at 216552_x_at 211688_x_at 208179_x_at 211687_x_at 207313_x_at216676_x_at 208198_x_at 210890_at), KIT (205051_s_at), KITLG(211124_s_at 226534_at 207029_at), KL (205978_at), KPNA2 (201088_at211762_s_at), KPNB1 (217027_x_at 208975_s_at 213507_s_at 208974_at),KRAS (1559204_at 1559203_s_at 204009_s_at 214352_s_at), KRT17 (212236_at205157_s_at), KRT18 (201596_at), KRT27 (240388_at), KRT7 (1558394_s_at209016_s_at), KRT8 (216568_at 230116_at 209008_at), KRTAP4-12(224269_at), LAIR1 (210644_s_at 208071_s_at), LAT (209881_s_at211005_at), LAX1 (207734_at), LCK (204891_s_at 204890_s_at), LCP2(205270_s_at 205269_at), LCT (206945_at), LDLRAP1 (221790_s_at57082_at), LEPR (209894_at 1556919_at 211356_at 211354_s_at 207255_at211355_at), LIME1 (219541_at), LMO4 (229537_at 241922_at 209204_at227155_at 209205_s_at), LMTK2 (235307_at 206223_at 226375_at), LMTK3(1557103_a_at), LPHN1 (219145_at 47560_at 203488_at), LPXN (216250_s_at242778_at), LRP1 (200785_s_at 1555353_at 200784_s_at), LRPPRC(211615_s_at 211971_s_at 230194_at 1557360_at 230594_at), LRSAM1(227675_at 235449_at), LTK (207106_s_at 217184_s_at), LYN (202625_at210754_s_at 202626_s_at), MAD2L1 (1554768_a_at 203362_s_at), MAG(217447_at 216617_s_at). MAGED1 (209014_at 244878_at), MAP2 (241044_at210015_s_at 225540_at), MAP2K1 (202670_at), MAP3K1 (225927_at214786_at). MAP3K14 (205192_at), MAP3K3 (227131_at 203514_at), MAP3K4(204089_at 216199_s_at), MAP3K5 (203837_at 203836_s_at), MAP4K1(214339_s_at 214219_x_at 206296_x_at), MAP4K5 (211081_s_at 203553_s_at203552_at), MAPK1 (208351_s_at 224621_at 1552264_a_at 1552263_at229847_at 212271_at), MAPK14 (202530_at 210449_at 211561_x_at211087_at), MAPK15 (241357_at), MAPK3 (212046_at), MAPK8 (226046_at226048_at 210671_x_at 210477_x_at), MAPK81P1 (213014_at 213013_at),MAPK81P2 (208603_s_at 205050_s_at), MAPK81P3 (216139_s_at 213177_at230162_s_at 213178_s_at 232085_at 216137_s_at), MAPK9 (210570_x_at225781_at 203218_at), MAPT (203929_s_at 203930_s_at 225379_at206401_s_at 203928_x_at), MATK (206267_s_at), MAX (209331_s_at209332_s_at 210734_at 208403_x_at 214108_at), MCAM (210869_s_at209086_x_at 211340_s_at 209087_x_at), MCF2 (1555313_a_at 217004_s_at208017_s_at), MDK (209035_at), MDM2 (217542_at 229711_s_at 225160_at217373_at 244616_at 205386_s_at 211832_s_at), MERTK (211913_s_at206028_s_at), MET (213807_x_at 203510_at 211599_x_at 213816_s_at),MICAL1 (218376_s_at), CLNK (1570239_a_at 1562587_at), MLLT4 (238871_at230622_at 208512_s_at), MME (203434_s_at 203435_s_at), MMP16 (207012_at223614_at 208167_s_at 208166_at 207013_s_at), MMP2 (201069_at), MPDZ(205079_s_at 213306_at), MPL (211903_s_at 216825_s_at 207550_at), MPZL1(210594_x_at 201874_at 210210_at 201875_s_at 210087_s_at 231621_at),MS4A1 (228592_at 228599_at 210356_x_at 217418_x_at), MS4A2 (207497_s_at207496_at), MSN (200600_at 240960_at), MST1 (216320_x_at 205614_x_at213380_x_at), MST1R (205455_at), MUC1 (211695_x_at 207847_s_at213693_s_at), MUC20 (231941_s_at 230043_at 226622_at), MUC4 (204895_x_at235055_at 217109_at 217110_s_at), MUSK (207632_at 207633_s_at241122_s_at), MYC (202431_s_at), MYD88 (209124_at), MYLK (1568770_at1563466_at 224823_at 202555_s_at), NCAM1 (214952_at 209968_s_at229799_s_at 227394_at 217359_s_at 212843_at), NCF1 (204961_s_at214084_at), NCK1 (211063_s_at 204725_s_at 229895_s_at), NCK2(203315_at), NCOA3 (207700_s_at 209061_at 211352_s_at 209062_x_at209060_x_at), NCOA6 (208979_at 1568874_at), NCOR1 (234313_at 200854_at200856_x_at 200855_at 200857_s_at), NCSTN (237076_at 208759_at), ND2(not_found), NDUFS1 (203039_s_at 229647_at 236356_at), NDUFS6(203606_at), NEDD4 (213012_at), NEDD4L (226974_at 212445_s_at 241396_at212448_at 236490_at), NEDD8 (243733_at 201840_at), NEDD9 (1569020_at202150_s_at 202149_at), NEO1 (204321_at 229877_at 225270_at), NF2(204991_s_at 238618_at 217150_s_at 211091_s_at 210767_at 218915_at211092_s_at 211017_s_at), NFAM1 (243099_at 230322_at), NFKBIA(201502_s_at), NFKBIB (214448_x_at 228388_at 214062_at), NGEF (227240_at243556_at), NGF (206814_at), NGFR (205858_at), NGFRAP1 (217963_s_at),NMT1 (201159_s_at 201157_s_at 201158_at), NOS2 (210037_s_at), NOTCH1(218902_at), NPHP1 (206285_at 238844_s_at 238843_at), NPHS1 (207673_at241181_at), NPM1 (200063_s_at 221923_s_at 221691_at), NR3C1 (201866_s_at232431_at 216321_s_at 201865_at 211671_s_at), NRAS (224985_at202647_s_at), NRG1 (208231_at 208232_x_at 208241_at 208230_s_at206237_s_at 206343_s_at), NRG2 (242303_at 208062_s_at 206879_s_at), NRG3(229233_at), NRG4 (242426_at), NRP1 (210510_s_at 212298_at), NRP2(223510_at 211844_s_at 210842_at 229225_at 1555468_at 230410_at210841_s_at 214632_at 228102_at 225566_at 228103_s_at), NRTN(210683_at), NTF3 (206706_at), NTF4 (not_found), NTRK1 (208605_s_at),NTRK2 (214680_at 207152_at 236095_at 221796_at 229463_at 221795_at),NTRK3 (217033_x_at 217377_x_at 215115_x_at 215025_at 206462_s_at1557795_s_at 228849_at), NUMB (209073_s_at 230462_at 207545_s_at), NUMBL(242195_at 224059_s_at), NXF1 (208922_s_at), OCLN (235937_at 209925_at),ODF2L (230926_s_at 237420_at 228577_at), ONECUT1 (210745_at), OSMR(1554008_at 226621_at 205729_at), P2RY2 (206277_at), PA2G4 (214794_at208676_s_at), PAG1 (225626_at 227354_at 225622_at), PAK1 (226507_at209615_s_at), PAK2 (208878_s_at 208877_at 208876_s_at 236283_at208875_s_at 205962_at 1559052_s_at 244268_x_at), PAK-4 (203154_s_at33814_at 215326_at), PARD3 (221526_x_at 221527_s_at 221280_s_at210094_s_at), PAX3 (207679_at 231490_at 231666_at 216059_at 207680_at),NAMPT (217739_s_at 217738_at 1555167_s_at), PCBD2 (1554894_a_at223712_at), PDAP1 (202290_at 217624_at), PDE4A (211901_s_at 211591_s_at204735_at 211447_s_at), PDE4D (228962_at 1554717_a_at 243586_at204491_at 222322_at 210837_s_at 210836_x_at 211840_s_at), PDE6G(210060_at), PDGFA (229830_at 216867_s_at 205463_s_at), PDGFB(204200_s_at 216061_x_at 217112_at), PDGFC (242171_at 222719_s_at218718_at), PDGFD (222860_s_at 219304_s_at), PDGFRA (211533_at 237696_at1554828_at 203131_at 215305_at), PDGFRB (202273_at), PDLIM7 (203369_x_at214266_s_at 214121_x_at 203370_s_at 214122_at), PDPK1 (221244_s_at232050_at 204524_at 32029_at 224986_s_at 222260_at), PECAM1 (1558397_at208983_s_at 208982_at 208981_at), PELP1 (215354_s_at), PGF(209652_s_at), PGR (208305_at 228554_at), PIAS1 (217864_s_at 222371_at),PICK1 (204746_s_at), PIK3AP1 (1554508_at 226459_at), PIK3C2A(1569022_a_at 235792_x_at 1553694_a_at 213070_at 1569021_at 226094_at),PIK3C2B (204484_at), PIK3CA (204369_at), PIK3CB (212688_at 217620_s_at),PIK3CG (206369_s_at 206370_at), PIK3R1 (212240_s_at 212249_at212239_at), PIK3R2 (229392_s_at 207105_s_at 1568629_s_at), PIK3R3(202743_at 211580_s_at), PILRB (225321_s_at 220954_s_at), PIM1(209193_at), PIP4K2A (205570_at 229713_at 212829_at), PIP4K2B(1553047_at 201081_s_at 1553048_a_at 201080_at), PIP4K2C (218942_at),PIP5K1A (210256_s_at 207391_s_at 211205_x_at), PIP5KIB (205632_s_at),PIPSKIC (212518_at), PITPNA (241974_at 201191_at 239124_at 201192_s_at201190_s_at 237424_at), PITPNM1 (203826_s_at). PITPNM2 (1552923_a_at1552924_a_at 232950_s_at), PITPNM3 (221254_s_at 230076_at), PKD1(216949_s_at 202327_s_at 202328_s_at), PKIA (204612_at), PLA2G4A(210145_at), PLAUR (210845_s_at 214866_at 211924_s_at), PLCE1 (205112_at205111_s_at), PLCG1 (216551_x_at 202789_at), PLCG2 (204613_at), PLD1(215723_s_at 226636_at 177_at 205203_at 1557126_a_at 215724_at). PLD2(209643_s_at), PLEC (216971_s_at 201373_at 238083_at), PLK4 (204887_s_at204886_at 211088_s_at), PLSCR1 (202446_s_at 202430_s_at), PLXNA1(1558140_at 221537_at 221538_s_at), PLXNB1 (215807_s_at 215668_s_at),PMAIP1 (204286_s_at 204285_s_at). POLA2 (204441_s_at), POLR2A (202725_at217420_s_at 217415_at), POLR2I (212955_s_at). POU2AF1 (205267_at), PPARD(210636_at 37152_at 208044_s_at), PPIA (217602_at 211378_x_at211978_x_at 211765_at 201293_x_at 226336_at 212661_at), PPP1CB(201409_s_at 201408_at 201407_s_at 228222_at), PPP1R15A (37028_at202014_at), PPP1R2 (202165_at 202166_s_at 213774_s_at), PPP1R8(207830_s_at), PPP2CA (208652_at), PPP2R1B (202886_s_at 202885_s_at202883_s_at 202884_s_at 222351_at), PPP2R5A (202187_s_at), PRAM1(241742_at), PRDX2 (215067_at 39729_at), PRKACA (202801_at 216234_s_at),PRKAR1A (242482_at 200604_s_at 200605_s_at 200603_at), PRKAR2A(204842_x_at 204843_s_at), PRKCA (213093_at 1560074_at 215195_at206923_at 215194_at), PRKCB (227817_at 209685_s_at 207957_s_at230437_s_at 228795_at 227824_at), PRKCD (202545_at), PRKCE (206248_at226101_at), PRKCH (218764_at 206099_at), PRKC1 (213518_at 209678_s_at209677_at), PRKCQ (210039_s_at 210038_at), PRKCZ (202178_at), PRKD1(205880_at 217705_at), PRKDC (208694_at 210543_s_at), PRLR (211917_s_at231981_at 243755_at 216638_s_at 227629_at 210476_s_at 206346_at), PRMT5(1564520_s_at 1564521_at 217786_at), PRO51 (207808_s_at), PSMA4(203396_at), PSMD13 (201233_at 201232_s_at), PSTPIP1 (211178_s_at),PTAFR (206278_at 211661_x_at 227184_at), PTEN (233314_at 233254_at211711_s_at 225363_at 227469_at 217492_s_at 204054_at 204053_x_at),PTGES3 (200627_at), PTK2 (1559529_at 208820_at 207821_s_at 241453_at),PTK2B (203110_at 203111_s_at), PTK6 (1553114_a_at), PTK7 (1555324_at207011_s_at), PTN (211737_x_at 209466_x_at 209465_x_at), PTPN1(202716_at 240260_at 239526_x_at 217689_at), PTPN11 (209895_at209896_s_at 205867_at 212610_at 205868_s_at 241930_at 1552637_at),PTPN12 (216915_s_at 202006_at 216884_at 244356_at), PTPN18 (203555_at213521_at 1569552_at), PTPN2 (213136_at 204935_at 213137_s_at), PTPN21(205438_at 226380_at 222092_at 40524_at 1320_at), PTPN22 (236539_at208010_s_at 206060_s_at), PTPN3 (227944_at 203997_at), PTPN5 (236456_at233471_at), PTPN6 (206687_s_at), PTPRA (213799_s_at 213795_s_at), PTPRB(205846_at 217177_s_at 230250_at), PTPRC (212587_s_at 207238_s_at1552480_s_at 1569830_at 212588_at), PTPRE (221840_at 1559018_at), PTPRF(215066_at 200637_s_at 200636_s_at 200635_s_at), PTPRH (208300_at),PTPRJ (210173_at 214137_at 227396_at), PTPRO (208121_s_at 1554199_at),PTPRS (1555666_at 210823_s_at 229465_s_at 226571_s_at), PTPRU(211320_s_at), PTPRZ1 (204469_at), PXN (211823_s_at 201087_at), RAD51(205023_at 205024_s_at), RAD52 (205647_at 210630_s_at 211904_at), RAD9A(204828_at 1562022_s_at), RADIL (223693_s_at), RAF1 (201244_s_at), RALA(214435_at 224880_at), RALB (202101_s_at 202100_at), RALGDS (209050_s_at209051_s_at), RAN (200750_s_at), RANBP10 (53987_at 221809_at1558773_s_at), RANBP9 (202582_s_at 216125_s_at 202583_s_at), RAP1A(1555339_at 202362_at 1555340_x_at), RAPGEF1 (225738_at 226389_s_at204543_at), RAPSN (211570_s_at), RASA1 (210621_s_at 202677_at), RASA3(225562_at 206221_at 206220_s_at), RASA4 (212706_at 208534_s_at212707_s_at), RASGEF1C (236748_at), RASGRF1 (214905_at 215688_at1554992_at 210550_s_at), RB1 (203132_at 211540_s_at), RB1CC1(202033_s_at 237626_at 202034_x_at), RELA (230202_at 201783_s_at209878_s_at), RET (215771_x_at 211421_s_at 205879_at), RFX1 (206321_at226786_at), RGS16 (209324_s_at 209325_s_at), RGS2 (202388_at), RGS4(204337_at 204339_s_at 204338_s_at), ARHGAP32 (242196_at 203431_s_at210791_s_at 229648_at), RIN1 (205211_s_at), RIN2 (209684_at 233811_at),RIPK1 (209941_at 226551_at), RITZ (239843_at 243463_s_at 209882_at),RIT2 (206984_s_at), RNF130 (217865_at), RNF41 (201961_s_at 201962_s_at),ROBO1 (213194_at 240558_at), ROCK1 (235854_x_at 214578_s_at 230239_at213044_at), ROR1 (205805_s_at 211057_at), ROR2 (231000_at 205578_at),ROS1 (207569_at), RPL10 (200724_at 221989_at 200725_at), RPL18A(200869_at), RPL8 (200936_at), RPLP2 (200908_s_at 200909_s_at), RPN1(201011_at), RPS6KA1 (203379_at), RPS6KA2 (240720_at 236658_at 212912_at204906_at 1557970_s_at), RRAS (212647_at), RTN1 (210222_s_at 203485_at),RTN3 (219549_s_at 224564_s_at), RUFY1 (218243_at 233380_s_at), RUFY2(1554133_at 238550_at 1569630_a_at 233192_s_at 235345_at), RUNX1(236114_at 209359_x_at 211182_x_at 211181_x_at 209360_s_at 210805_x_at211180_x_at 208129_x_at 211620_x_at 210365_at), RUSC1 (206949_s_at),RYBP (201846_s_at 242719_at 201844_s_at 237456_at 201845_s_at), RYK(214172_at 202853_s_at 216976_s_at 238210_at), S100A7 (205916_at),S100A9 (203535_at), SAT1 (210592_s_at 213988s_at 203455_s_at), SCAMP1(212425_at 1552978_a_at 206668_s_at 212416_at 206667_s_at 212417_at),SCAMP3 (201771_at), SDC2 (212154_at 212158_at 212157_at), SDC3(1554864_a_at 202898_at), SDCBP (200958_s_at), SEC13 (207707_s_at), SELE(206211_at), SELPLG (209879_at 209880_s_at), SERPINA3 (202376_at), SEZ6(243430_at 229651_at), SF3B3 (200688_at 200687_s_at), SF3B4 (209044_at),SFN (33323_r_at 33322_i_at 209260_at), SRSF5 (210077_s_at 203380_at212266_s_at), SGSM2 (212319_at 36129_at), SH2B1 (209322_s_at 40149_at),SH2B2 (205367_at), SH2B3 (203320_at), SH2D1A (211210_x_at 211209_at211211_at 210116_at), SH2D1B (1553177_at 1553176_at), SH2D2A(207351_s_at), SH2D3A (219513_s_at 222169_at), SH2D3C (226673_at1552667_a_at), SH3BGRL (201312_s_at 201311_s_at), SH3BGRL3(221269_s_at), SH3BP1 (215799_at 213633_at), SH3BP2 (211250_s_at209370_s_at 209371_s_at), SH3BP5 (201811_x_at 201810_s_at), SH3GL2(205751_at), SH3 KBP1 (1554168_a_at 235692_at 223082_at), SH3PXD2A(224817_at 213252_at 207661_s_at), SHB (204657_s_at 230459_s_at204656_at 1557458_s_at 243595_at), SHC1 (201469_s_at 214853_s_at), SHC2(213464_at), SHC3 (243881_at 206330_s_at 229824_at), SHD (227845_s_at),SHE (229910_at), SHF (228922_at), SIRPA (202897_at 202896_s_at202895_s_at 217024_x_at), SITZ (205484_at), SIVA1 (210792_x_at203489_at), SKAP1 (205790_at), SKAP2 (216899_s_at 204362_at 204361_s_at225639_at), SLA (203761_at 203760_s_at), SLA2 (232234_at 1555688_s_at),SLAMF1 (1555626_a_at 206181_at), SLAMF6 (1552497_a_at), SLC25A6(212085_at 212826_s_at), SLC2A1 (235633_at 201250_s_at 201249_at),SLC3A2 (200924_s_at), SLC4A1 (205592_at 1552713_a_at), SLC9A2(211116_at), SLC9A3R1 (201349_at), SLC9A3R2 (209830_s_at), SMN1(242495_at 203852_s_at), SMAD2 (239271_at 226563_at 243895_x_at203076_s_at 203075_at 235598_at 203077_s_at), SMAD4 (1565702_at202527_s_at 235725_at 202526_at 235622_at), SMARCB1 (212167_s_at),SMARCE1 (229511_at 211989_at 211988_at), SMC1A (1555677_s_at 239688_at217555_at 201589_at), SMG7 (242575_at 217189_s_at 201793_at201794_s_at), SMURF2 (232020_at 230820_at 227489_at 205596_s_at), SNAP29(239084_at 222597_at 218327_s_at), SNAPIN (223066_at), SNCA (211546_x_at207827_at 236081_at 204467_s_at 204466_s_at), SNRPD2 (200826_at), SNTB2(227312_at 226685_at 213814_s_at 205314_at 238925_at 205315_s_at), SNURF(206042_at 201522_at), SNX1 (213364_s_at 201716_at 214531_s_at216357_at), SNX2 (202113_s_at 202114_at), ARHGAP33 (233885_at 215256_at213827_at), SNX4 (205329_s_at 212652_s_at), SNX6 (217789_at222410_s_at), SNX9 (223028_s_at 223027_at), SOCS1 (210001_s_at 209999_at210000_s_at 213337_s_at), SOCS2 (203373_at 203372_s_at), SOCS3(214105_at 206360_s_at 206359_at 227697_at), SOCS5 (208127_s_at209648_at 209647_s_at), SOCS6 (227542_at 206020_at 214462_at), SORBS1(237026_at 211705_s_at 211819_s_at 222513_s_at 218087_s_at), SORBS2(1558815_at 237285_at 241104_at 225728_at 240120_at 204288_s_at220858_at), SOS1 (1557354_at 212777_at 242018_at 242682_at 229261_at227426_at 212780_at), SOS2 (217575_s_at 217644_s_at 217576_at 212870_at211665_s_at), SPHK1 (219257_s_at), SPHK2 (209857_s_at 40273_at), SPN(216980_s_at 216981_x_at 206057_at 206056_x_at 1568964_x_at), SPRED1(244439_at 235074_at 226837_at), SPRED2 (212466_at 212458_at214026_s_at), SPSB1 (226075_at 219677_at), SPTAN1 (214926_at 215235_at214925_s_at 208611_s_at), SQSTM1 (217252_at 244804_at 201471_s_at217255_at 213112_s_at), SRC (1565082_at 237103_at 213324_at 1565080_at221284_s_at 1558211_s_at), SRF (202401_s_at 202400_s_at), SRM(201516_at), ST5 (202440_s_at), STAM (203544_s_at), STAM2 (228254_at215044_s_at 208194_s_at 242569_at 209649_at), STAP1 (220059_at1554343_a_at), STAP2 (221610_s_at), STAT1 (AFFX-HUMISGF3AM97935_(—)5_atAFFX-HUMISGF3AM979353_at 200887_s_at AFFX-HUMISGF3AM97935_MB_atAFFX-HUMISGF3AM97935_MA at 209969_s_at), STAT2 (225636_at 205170_at217199_s_at), STAT3 (208991_at 225289_at 208992_s_at 243213_at), STAT5A(203010_at), STAT5B (1555086_at 212550_at 205026_at 1555088_at212549_at), STAT6 (201332_s_at 201331_s_at), STK39 (202786_at), STUB1(217934_x_at 233049_x_at 227625_s_at), SUPT4H1 (201484_at 201483_s_at),SUPT6H (208830_s_at 208420_x_at 1554311_a_at 208831_at), SYK (226068_at209269_s_at 207540_s_at 244023_at), SYN1 (221914_at 1553264_a_at),SYNCRIP (217834_s_at 209025_s_at 209024_s_at 217833_at 217832_at1555427_s_at), SYNE1 (244070_at 215350_at 209447_at 244144_at), SYNGAP1(230297_x_at 234285_at), SYNJ1 (212990_at 232993_at 207594_s_at), TAF1(227205_at 216955_at 216711_s_at), TEC (206301_at), TEK (217711_at206702_at), TENC1 (212494_at), TERT (207199_at 1555271_a_at), TGFA(211258_s_at 205016_at 205015_s_at), TGFB1I1 (209651_at), TGFBR1(224793_s_at 206943_at), TH1L (225006_at 225865_x_at 225261_at220607_x_at), THOC5 (209418_s_at 209419_at). THY1 (213869_x_at208850_s_at 208851_s_at), TIAF1 (202039_at), TIAM1 (213135_at206409_at), TIE1 (1560657_at 204468_s_at), TIMP3 (201149_s_at201150_s_at 201147_s_at 201148_s_at), TIRAP (1554091_a_at 239796_x_at236687_at 1552360_a_at 1552804_a_at), TJP1 (214168_s_at 202011_at), TLN1(203254_s_at), TLR2 (204924_at), TLR3 (206271_at 239587_at), TLR4(221060_s_at 1552798_a_at 224341_at 232068_s_at), TLR6 (239021_at207446_at), TLR8 (220832_at 229560_at), TLR9 (223903_at), TM4SF1(209387_s_at 238168_at 209386_at 215033_at 215034_s_at), TMF1(215855_s_at 227685_at 214948_s_at 235566_at 213024_at), TNC(201645_at), TNFAIP1 (201208_s_at 201207_at), TNFRSF10A (1552648_a_at),TNFRSF11A (207037_at 238846_at), TNFRSF1A (207643_s_at), TNFRSF1B(203508_at), TNFRSF8 (206729_at), TNFSF11 (211153_s_at 241248_at210643_at), TNIP2 (48531_at 218335_at 232160_s_at), TNK1 (217149_at205793_x_at), TNK2 (203839_s_at 216439_at 1555557_a_at 228279_s_at203838_s_at), TNS1 (221748_s_at 218863_s_at 221246_at 221747_at218864_at), TNS4 (230398_at 222265_at), TOB1 (228834_at 202704_at),TOM1L1 (240261_at 204485_s_at), TP53 (211300_s_at 201746_at), TP53BP2(203120_at), TP53RK (225402_at 235192_at). TP73 (1554379_a_at 232546_at220804_s_at), TRAF1 (235116_at 205599_at), TRAF2 (204413_at), TRAF3(208315_x_at 221571_at), TRAF6 (205558_at), TRAT1 (217147_s_at), TR10(240399_at 209010_s_at 209011_at 209012_at 244527_at 209013_at 240773_at208178_at), TRIP6 (209129_at), TRPC6 (217287_s_at 241558_at 206528_at),TRPV4 (219516_at), TSG101 (230176_at 201758_at). TSHR (215443_at215442_s_at 210055_at), TTR (209660_at), TUB (208431_s_at 228882_at210737_at), TUBA1A (211072_x_at 211058_x_at 201090_x_at 209118_s_at211750_at 212639_at 209251_at 213646_x_at), TUBA1B (211072_x_at211058_at 201090_at 211750_at 212639_at 209251_x_at 213646_x_at), TUBA3C(210527_x_at 216323_x_at), TUBA4A (212242_at), TUBB (212320_at211714_x_at 209026_x_at), TUBB2A (213476_at 204141_at 209372_x_at), TXK(206828_at), TYK2 (205546_s_at), TYRO3 (211432_s_at 211431_s_at1566934_at), TYROBP (204122_at), UBASH3B (244068_at 240387_at 238587_at238462_at 228353_at 228359_at), UBB (200633_at 242300_at), UBE2L3(200684_s_at 200683_s_at 200682_s_at 200676_s_at), UBE3A (211575_s_at213128_s_at 211285_s_at 213291_s_at 212278_at), UCP2 (208998_at208997_s_at), UNC119 (203271_s_at), UQCRB (209066_x_at 209065_at205849_s_at 244293_at), VAV1 (206219_s_at), VAV2 (205537_s_at 226063_at205536_at), VAV3 (218806_s_at 224221_s_at 218807_at), VCL (200930_s_at200931_s_at), VCP (208649_s_at 228442_at 208648_at), VDR (204255_s_at204254_s_at 204253_s_at 213692_s_at), VEGFA (212171_at 211527_at210512_s_at 210513_s_at), VEGFB (203683_s_at), VEGFC (209946_at), VIL1(1554943_at 1554945_x_at 205506_at), VTI1B (209452_s_at 225926_at), WAS(205400_at 38964_r_at). WASF1 (204165_at), WASF2 (224563_at 224562_at221725_at), WASL (224813_at 205810_s_at 230340_s_at 205809_s_at), WBP11(217822_at 217821_s_at), SNRNP40 (215905_s_at), WIPF1 (202664_at231182_at 202665_s_at 202663_at), WISP2 (205792_at), WNT1 (208570_at),WNT3A (not_found), WNT5A (213425_at 205990_s_at 231227_at), WT1(216953_s_at 206067_s_at), WWOX (237035_at 223868_s_at 219077_s_at221147_x_at 210695_s_at 242099_at 223747_at), XPO1 (208775_at), XPO6(211982_x_at 214784_at), XRCC5 (208643_s_at 208642_s_at), XRCC6(200792_at), YAP1 (224895_at 224894_at 213342_at), YES1 (202932_at202933_s_at).

Example 5 Proximity Ligation Assay for Egfr and Grb2 Interaction in CellLines Using Intracellular EGFR Epitope Updated Methods ReagentInformation: Antibodies:

mGRb2 (Mouse monoclonal, clone 81 (Cat#610112, BD Biosciences)

rEGFR (Rabbit monoclonal, clone D38B1 (Cat#4267, Cell Signaling)

Working dilutions: m-EGFR (1:50)+r-GRb2 (1:50)

Solutions:

_(MQ)H₂O

PBS

Kit Components:

Secondary antibody-PLA probes (plus and minus)

Ligation reagents

Amplification ligation reagents

1× Wash Buffer B

0.01× Wash Buffer B

Mounting media (wDAPI)

Experimental Procedure:

1. Plate cells in chamber wells 24 h before processing. For most celllines, 50×10⁴ per well (0.8 cm²) is sufficient. This number should beadjusted for longer culture times and different-sized chamber wells.

2. Wash each well with PBS (2×2 m).

3. Fix cells by adding 400 μl 10% Buffered formalin per well. Incubate20 m RT with gentle rocking.

4. Wash each well with PBS (3×2 m).

5. Pennabilize cells by adding 0.5% Triton X100 (diluted in PBS).Incubate 10 m RT, no rocking.

6. Wash each well with PBS (3×2 m).

7. Remove chamber from slide. Note that excess silicone adhesive shouldbe removed with a scalpel to ensure that it does not interfere withcoverslipping later.

8. Add 1.5% BSA (diluted in PBS) using a volume sufficient to fullycover each well. For 8-well slides (0.8 cm²), a volume of 50 μl issufficient. A grease pen can be used to ensure a hydrophopic barrierexists between wells, but is usually not necessary. Incubate 30 m RT inhumidity chamber.

9. Primary antibodies: Add 50 μl per well appropriate primary antibodysoultuion [r-EGFR and m-GRb2] diluted in PBS. Incubate ON in humiditychamber 4° C. with gentle rocking.

a. Record start time

b. Incubation time should be ˜16-18 hours

10. Next day: Tap off primary antibody solution. Wash 2 times withsequential immersion in PBS in Coplin jars. Wash 2×5 m RT with gentlerocking.

11. PLA probes (secondary antibody-DNA conjugates): Mix and dilute thetwo PLA probes 1:5 (diluted in PBS) (use 8 μl minus probe and 8 μl probeplus and 24 μl PBS=40 μl cm²). Ensure complete coverage of tissue area.Incubate slides 1 h 37° C. in pre-heated humidity chamber.

12. Tap off PLA probes and wash. Wash 2×5 m with PBS, RT with gentlerocking. Thaw 5× ligation mix at beginning of washes.

13. PLA probe ligation: Dilute ligation stock 1:5 in _(MQ)H₂O whilewaiting, saving room for ligase (e.g., 8 μl 5× ligation mix and 31 μl_(MQ)H₂O=39 μl). Add Ligase into ligation solution 1:40 during finalwash above. Cover tissue area completely using 40 μl/cm². Incubate for30 m 37° C. in pre-warmed humidity chamber.

14. Tap off ligation solution and wash. Wash 2×2 m RT with gentlerocking (PBS). Thaw 5× amplification mix at beginning of washes(avoiding light).

15. PLA probe amplification-hybridization-fluorescence: DiluteAmplification stock 1:5 in MQH2O while waiting (8 μl and 31.5 μl_(MQ)H₂O=39 μl). Add Polymerase 1:80 into amplification solution thenadd polymerase-amplification solution. Cover tissue area completelyusing 40 μl/cm². Incubate 2 h 37° C. pre-warmed humidity chamber (avoidlight).

16. Tap off amplification solution and wash 2×10 m RT with 1× WashBuffer B. Rinse slide in 0.01× Wash Buffer B.

17. Allow slides to dry for ˜10 m in dark. Mount with Invitrogen ProlongGold with DAPI. Add mounting media to cover slip and gently press downonto tissue area. Allow to dry overnight and image by confocalmicroscopy next day. Note that Prolong Gold is an aqueous-based mountingmedia and will not cure. Care should be taken to avoid moving coverslip,once mounted. Store slides at 4° after initial overnight dry.

18. Confocal Microscopy: Use the Leica TCS SP5 laser scanning confocalmicroscope. Place slide on scope and find best in-focus plane. Take careto avoid imaging along edges of tissue, whenever possible. Note that Cy5foci cannot be seen through the confocal eyepiece due to the far redwavelength. Use 40× oil objective and begin with voltage settings of˜450 for the 405 laser line (DAPI) and 580 for the 633 laser line (Cy5).Use 1024×1024 image size and acquire at 200 Hz.

19. Use the “live” scan to observe foci. Use the z-plane wheel to scrollabove and below the focus plane until foci disappear and only DAPIremains, set upper and lower limits. Generally, 12 z-slices at 0.76 μmprovides sufficient coverage of all foci.

20. Do not adjust 633 laser line between samples. DAPI can be adjustedand will vary among cell types. Keep DAPI relatively dim as the mergedimages may be overexposed if DAPI is too intense.

21. After collecting samples (2-3 fields of view for each sample),process each series as a maximum projection. These images can then beexported as TIFF files.

Example 6 Duolink Assay for EGFR and Grb2 Interaction from FFPE Tissueswith Cytokeratin Counterstaining Reagent Information Antibodies:

GRB2 (Mouse monoclonal, clone 81 (Cat#610112, BD Biosciences)

EGFR (Rabbit monoclonal, clone D38B1 (Cat#4267, Cell Signaling)

Cytokeratin, pan (Guinea Pig polyclonal (Cat#9 9097-48B 097-48B, USBiological)

AlexFluor555, goat anti-guinea pig (Invitrogen, Cat#A-21435)

Working dilutions: m-EGFR (1:100)+r-GRb2 (1:100)+gp-CK (1:100)

Solutions:

AR Buffer (PT4) (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH9)

PBS

_(MQ)H₂O

0.05% PBST (1 L PBS+5004 Tween20)

Kit Components:

Secondary antibody-PLA probes (plus and minus)

Ligation reagents

Amplification ligation reagents

1× Wash Buffer B

0.01× Wash Buffer B

Mounting media (wDAPI)

Experimental Procedure:

1. Antigen Retrieval: DeparrafinizeRehydrate slides. Perform successivewashes in Xylenes and ethanol gradient as follows:

a. 10 m Xylene

b. 10 m Xylene (use fresh xylene for this wash, can rotate forward)

c. 10 m 50:50 Xylene:Ethanol

d. 5 m 100% ethanol* Begin setup of pressure cooker here

e. 5 m 100% ethanol

f. 5 m 95% ethanol

g. 5 m 70% ethanol

h. 5 m 50% ethanol

i. H2O; can hold here if pressure cooker not ready

2. Antigen retrieval (HIER method, using pressure cooker). Bring 3 L of1×PT4 (TE, pH9) [Use a metal slide holder placed within an eppendorftube jar] to a low boil in pressure cooker. Place slide rack ineppendorf tube jar, seal and boil for 20 m. Remove from heat and letcool for 20 min.

3. Wash slide with PBS briefly and use grease pen to trace along edgesof TMA or tissue. Work as quickly as possible aspirating area aroundspots in order to delineate area without spots drying.

-   -   a. Record approximate area in cm²

4. Rinse briefly in PBST (0.05%) and add 1.5% BSA using a volumesufficient to fully cover delineated area. Ensure that hydrophopicbarrier created by grease pen is maintained. Incubate 30 m RT inhumidity chamber.

5. Primary antibodies: Add 200-300 μl/slide appropriate primaryantibodies [r-EGFR and m-GRb2] in 0.15% BSA (diluted in 0.05% PBST)Incubate ON in humidity chamber 4° C. with gentle rocking.

-   -   a. Record start time    -   b. Incubation time should be ˜16-18 h

6. Next day: Tap off primary antibody solution. Wash 2 times withsequential immersion in 0.05% PBST in Coplin jars. Wash 2×5 m RT withgentle rocking (0.05% PBST).

7. PLA probes (secondary antibody-DNA conjugates): Mix and dilute thetwo PLA probes 1:5 in 0.15% BSA (diluted in 0.05% PBST) (use 8 μl minusprobe and 8 μl probe plus and 24 μl PBS=40 μl cm²). Ensure completecoverage of tissue area. Incubate slides 1 h 37° C. in pre-heatedhumidity chamber.

8. Tap off PLA probes and wash. Wash 2×5 m RT with gentle rocking (0.05%PBST). Thaw 5× ligation mix at beginning of washes.

9. PLA probe ligation: Dilute ligation stock 1:5 in _(MQ)H₂O whilewaiting, saving room for ligase (e.g., 8 μl 5× ligation mix and 31 μl_(MQ)H₂O=39 μl). Add Ligase into ligation solution 1:40 during finalwash above. Cover tissue area completely using 40 μl cm². Incubate for30 m 37° C. in pre-warmed humidity chamber.

10. Tap off ligation solution and wash. Wash 2×2 m RT with gentlerocking (0.05% PBST). Thaw 5× amplification mix at beginning of washes(avoiding light).

11. PLA probe amplification-hybridization-fluorescence: DiluteAmplification stock 1:5 in _(MQ)H₂O while waiting (8 μl and 31.50_(MQ)H₂O=390). Add Polymerase 1:80 into amplification solution then addpolymerase-amplification solution. Cover tissue area completely using.400 cm². Incubate 2 h 37° C. pre-warmed humidity chamber (avoid light).

12. Tap off amplification solution and rinse slides with PBS. Incubatewith antipan cytokeratin (diluted 1:100) in PBS. Incubate 1 h 37° C.

13. Tap of antibody solution, wash twice with PBS (2×5 m RT with gentlerocking).

14. Add Alexa-Fluor555, diluted 1:500 in PBS. Incubate 30 m at 37° C.

15. Tap off aantibody solution and wash 2×10 m RT with 1× Wash Buffer B.Rinse slide in 0.01× Wash Buffer B.

16. Allow slides to dry for ˜10 m in dark. Mount with Invitrogen ProlongGold with DAPI. Add mounting media to cover slip and gently press downonto tissue area. Allow to dry overnight and image by confocalmicroscopy next day. Note that Prolong Gold is an aqueous-based mountingmedia and will not cure. Care should be taken to avoid moving coverslip,once mounted. Store slides at 4° after initial overnight dry.

17. Confocal Microscopy: Use the Leica TCS SP5 laser scanning confocalmicroscope. Place slide on scope and find best in-focus plane. Take careto avoid imaging along edges of tissue, whenever possible. Note that Cy5foci cannot be seen through the confocal eyepiece due to the far redwavelength. Use 40× oil objective and begin with voltage settings of˜450 for the 405 laser line (DAPI), ˜600 for the 543 laser line (Cy3)and ˜580 for the 633 laser line (Cy5). Use 1024×1024 image size andacquire at 200 Hz.

18. Use the “live” scan to observe foci. Use the z-plane wheel to scrollabove and below the focus plane until foci disappear and only DAPIremains, set upper and lower limits. Generally, 12 z-slices at 0.76 μmprovides sufficient coverage of all foci.

19. Do not adjust 633 laser line between samples. DAPI and Cy3 can beadjusted and will vary among cell types. Keep DAPI relatively dim as themerged images may be overexposed if DAPI is too intense.

20. After collecting samples (2-3 fields of view for each sample),process each series as a maximum projection. These images can then beexported as TIFF files.

Example 7 Proximity Ligation Assay for Phosphorylated RTKS in Cell LinesAntibodies (Varies):

pEGFRY1068 (Rabbit monoclonal, clone D38B1 Cat#4267, Cell Signaling)

ALK (rabbit monoclonal, clone D5F3 Cat #3633, Cell Signaling)

P-Tyr-100 (mouse monoclonal, Cat#9411, Cell Signaling)

Working dilutions: pEGFRY1068 (1:50), ALK (1:50), P-Tyr-100 (1:1000)

Solutions:

_(MQ)H₂O

PBS

Kit components:

-   -   Secondary antibody-PLA probes (plus and minus)    -   Ligation reagents    -   Amplification ligation reagents    -   1× Wash Buffer B    -   0.01× Wash Buffer B    -   Mounting media (w DAPI)

Experimental Procedure:

1. Plate cells in chamber wells 24 h before processing. For most celllines, 50×10⁴ per well (0.8 cm²) is sufficient. This number should beadjusted for longer culture times and different-sized chamber wells.

2. Wash each well with PBS (2×2 m).

3. Fix cells by adding 400 μl 10% Buffered formalin per well. Incubate20 m RT with gentle rocking.

4. Wash each well with PBS (3×2 m).

5. Permabilize cells by adding 0.5% Triton X100 (diluted in PBS).Incubate 10 m RT, no rocking.

6. Wash each well with PBS (3×2 m).

7. Remove chamber from slide. Note that excess silicone adhesive shouldbe removed with a scalpel to ensure that it does not interfere withcoverslipping later.

8. Add 1.5% BSA (diluted in PBS) using a volume sufficient to fullycover each well. For 8-well slides (0.8 cm²), a volume of 50 μl issufficient. A grease pen can be used to ensure a hydrophopic barrierexists between wells, but is usually not necessary. Incubate 30 m RT inhumidity chamber.

9. Primary antibodies: Add 50 μl per well appropriate primary antibodysoultuion diluted in PBS. Here, can use either a single antibody ordouble (depends on epitope availability and goals of experiment).Incubate ON in humidity chamber 4° C. with gentle rocking.

-   -   a. Record start time    -   b. Incubation time should be ˜16-18 h

10. Next day: Tap off primary antibody solution. Wash 2 times withsequential immersion in PBS in Coplin jars. Wash 2×5 m RT with gentlerocking.

-   -   11. PLA probes (secondary antibody-DNA conjugates): Note: if        performing single recognition experiment ensure that a plus and        minus probe from the same species as primary antibody are used.        Mix and dilute the two PLA probes 1:5 (diluted in PBS) (use 8 μl        minus probe and 8 μl probe plus and 24 μl PBS=40 μl/cm²). Ensure        complete coverage of tissue area. Incubate slides 1 h 37° C. in        pre-heated humidity chamber.

12. Tap off PLA probes and wash. Wash 2×5 m with PBS, RT with gentlerocking. Thaw 5× ligation mix at beginning of washes.

13. PLA probe ligation: Dilute ligation stock 1:5 in _(MQ)H₂O whilewaiting, saving room for ligase (e.g., 8 μl 5× ligation mix and 31 μl_(MQ)H₂O=39 μl). Add Ligase into ligation solution 1:40 during finalwash above. Cover tissue area completely using 40 μl/cm². Incubate for30 m 37° C. in pre-warmed humidity chamber.

14. Tap off ligation solution and wash. Wash 2×2 m RT with gentlerocking (PBS). Thaw 5× amplification mix at beginning of washes(avoiding light).

15. PLA probe amplification-hybridization-fluorescence: DiluteAmplification stock 1:5 in _(MQ)H₂O while waiting (8 μl and 31.5 μl_(MQ)H₂O=39 μl). Add Polymerase 1:80 into amplification solution thenadd polymerase-amplification solution. Cover tissue area completelyusing 40 μl/cm². Incubate 2 h 37° C. pre-warmed humidity chamber (avoidlight).

16. Tap off amplification solution and wash 2×10 m RT with 1× WashBuffer B. Rinse slide in 0.01× Wash Buffer B.

17. Allow slides to dry for ˜10 m in dark. Mount with Invitrogen ProlongGold with DAPI. Add mounting media to cover slip and gently press downonto tissue area. Allow to dry overnight and image by confocalmicroscopy next day. Note that Prolong Gold is an aqueous-based mountingmedia and will not cure. Care should be taken to avoid moving coverslip,once mounted. Store slides at 4° after initial overnight dry.

18. Confocal Microscopy: Use the Leica TCS SP5 laser scanning confocalmicroscope. Place slide on scope and find best in-focus plane. Take careto avoid imaging along edges of tissue, whenever possible. Note that Cy5foci cannot be seen through the confocal eyepiece due to the far redwavelength. Use 40× oil objective and begin with voltage settings of˜450 for the 405 laser line (DAPI) and 580 for the 633 laser line (Cy5).Use 1024×10²⁴ image size and acquire at 200 Hz.

19. Use the “live” scan to observe foci. Use the z-plane wheel to scrollabove and below the focus plane until foci disappear and only DAPIremains, set upper and lower limits. Generally, 12 z-slices at 0.76 μmprovides sufficient coverage of all foci.

20. Do not adjust 633 laser line between samples. DAPI can be adjustedand will vary among cell types. Keep DAPI relatively dim as the mergedimages may be overexposed if DAPI is too intense.

21. After collecting samples (2-3 fields of view for each sample),process each series as a maximum projection. These images can then beexported as TIFF files.

Example 8 Dulink Assay for EGFR and Grb2 Interaction from FFPE Tissueswith Brightfield Detection Reagent Information: Antibodies:

GRB2 (Mouse monoclonal, clone 81 (Cat#610112, BD Biosciences)

EGFR (Rabbit monoclonal, clone D38B1 (Cat#4267, Cell Signaling)

Working dilutions: m-EGFR (1:100)+r-GRb2 (1:100)

Solutions:

AR Buffer (PT4) (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH9) PBS

_(MQ)H₂O

0.05% PBST (1 L PBS+5004 Tween20)

Kit Components:

Secondary antibody-PLA probes (plus and minus)

Ligation reagents

Amplification reagents

Detection reagents

Counterstain reagents

1× Wash Buffer B

0.01× Wash Buffer B

Mounting media (wDAPI)

Experimental Procedure:

21. Antigen Retrieval: DeparrafinizeRehydrate slides. Perform successivewashes in Xylenes and ethanol gradient as follows:

-   -   a. 10 m Xylene    -   b. 10 m Xylene (use fresh xylene for this wash, can rotate        forward)    -   c. 10 m 50:50 Xylene:Ethanol    -   d. 5 m 100% ethanol* Begin setup of pressure cooker here    -   e. 5 m 100% ethanol    -   f. 5 m 95% ethanol    -   g. 5 m 70% ethanol    -   h. 5 m 50% ethanol    -   i. H2O; can hold here if pressure cooker not ready

22. Antigen retrieval (HIER method, using pressure cooker). Bring 3 L of1×PT4 (TE, pH9) [Use a metal slide holder placed within an eppendorftube jar] to a low boil in pressure cooker. Place slide rack ineppendorf tube jar, seal and boil for 20 m. Remove from heat and letcool for 20 min.

23. Wash slide with PBS briefly and use grease pen to trace along edgesof TMA or tissue. Work as quickly as possible aspirating area aroundspots in order to delineate area without spots drying.

-   -   a. Record approximate area in cm²

24. Rinse briefly in PBST (0.05%) and

25. Add 1.5% BSA using a volume sufficient to fully cover delineatedarea. Ensure that hydrophopic barrier created by grease pen ismaintained. Incubate 30 m RT in humidity chamber.

26. Primary antibodies: Add 200-300 μ/slide appropriate primaryantibodies [r-EGFR and m-GRb2] in 0.15% BSA (diluted in 0.05% PBST)Incubate ON in humidity chamber 4° C. with gentle rocking.

-   -   a. Record start time    -   b. Incubation time should be ˜16-18 h

27. Next day: Tap off primary antibody solution. Wash 2 times withsequential immersion in 0.05% PBST in Coplin jars. Wash 2×5 m RT withgentle rocking (0.05% PBST).

28. PLA probes (secondary antibody-DNA conjugates): Mix and dilute thetwo PLA probes 1:5 in 0.15% BSA (diluted in 0.05% PBST) (use 80 minusprobe and 80 probe plus and 24 μl PBS=40 μl/cm²). Ensure completecoverage of tissue area. Incubate slides 1 h 37° C. in pre-heatedhumidity chamber.

29. Tap off PLA probes and wash. Wash 2×5 m RT with gentle rocking(0.05% PBST). Thaw 5× ligation mix at beginning of washes.

30. PLA probe ligation: Dilute ligation stock 1:5 in _(MQ)H₂O whilewaiting, saving room for ligase (e.g., 8 μl 5× ligation mix and 31 μl_(MQ)H₂O=39 μl). Add Ligase into ligation solution 1:40 during finalwash above. Cover tissue area completely using 40 μl cm². Incubate for30 m 37° C. in pre-warmed humidity chamber.

31. Tap off ligation solution and wash. Wash 2×2 m RT with gentlerocking (0.05% PBST). Thaw 5× amplification mix at beginning of washes.

32. PLA probe amplification: Dilute Amplification stock 1:5 in _(mQ)H₂Owhile waiting (8 μl and 31.5 μl _(MQ)H₂O=39 μl). Add Polymerase 1:80into amplification solution then add polymerase-amplification solution.Cover tissue area completely using 40 μl/cm². Incubate 2 h 37° C.pre-warmed humidity chamber.

33. Tap off amplification solution and wash. Wash 2×2 m RT with gentlerocking (0.05% PBST). Thaw 5× detection mix at beginning of washes(avoiding light).

34. Dilute 5× detection reagent in H₂0 and add to slides. Incubate 1 h37° C.

35. Rinse 2×5 m in PBST, then add detection reagents (A, B, C, D) andincubate 10 m.

36. Rinse 2×2 m in H₂O and add hematoxylin nuclei stain dropwise.Incubate 2 m RT. Rinse for 10 m under running tap water.

37. Dehydrate slides by incubating in increasing concentrations ofethanol and then incubate in two changes of xylene (reversal of step 1in the protocol). Allow slides to dry in fume hood.

38. Mount slides in toluene-based mounting media and allow to cureovernight.

39. Brightfield Microscopy: Observe slides under light microscopy at200×, 400×, 630× or 1000×. Alternatively, slides may be scanned on aslide scanner.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication. In addition, any elements or limitations of any inventionor embodiment thereof disclosed herein can be combined with any and/orall other elements or limitations (individually or in any combination)or any other invention or embodiment thereof disclosed herein, and allsuch combinations are contemplated with the scope of the inventionwithout limitation thereto.

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1. A method for assessing the sensitivity of a malignancy to a potentialtreatment, comprising comparing a protein-protein interaction (PPI)profile obtained from a sample of the malignancy to a reference PPIprofile.
 2. The method of claim 1, wherein the sample PPI profilerepresents the abundance of target binding partners that are inproximity to each other within the sample.
 3. The method of claim 2,wherein the sample PPI profile represents the abundance of two bindingpartners that are in proximity to each other within the sample.
 4. Themethod of claim 2, wherein the sample PPI profile represents theabundance of three or more binding partners that are in proximity toeach other within the sample.
 5. The method of claim 1, wherein thesample PPI profile represents the abundance of target binding partnersthat are in proximity to each other within the sample, and wherein atleast one of the target binding partners is a receptor tyrosine kinase(RTK).
 6. The method of claim 1, wherein at least one of the targetbinding partners is of an RTK class selected from among RTK class I (EGFreceptor family; ErbB family), RTK class II (insulin receptor family),RTK class III (PDGF receptor family), RTK class IV (FGF receptor family)RTK class V (VEGF receptors family), RTK class VI (HGF receptor family),RTK class VII (Trk receptor family), RTK class VIII (Eph receptorfamily), RTK class IX (AXL receptor family), RTK class X (LTK receptorfamily), RTK class XI (TIE receptor family), RTK class XII (ROR receptorfamily), XIII (DDR receptor family), RTK class XIV (RET receptorfamily), RTK class XV (KLG receptor family), RTK class XVI (RYK receptorfamily), and RTK class XVII (MuSK receptor family). 7-10. (canceled) 11.The method of claim 2, wherein the binding partners of the PPI and thedownstream effectors of the PPI do not harbor any sequence mutationsknown to be associated with sensitivity to a treatment such as a ppimodulator.
 12. The method of claim 2, wherein the sample PPI profile andthe reference PPI profile are each expressed as a value representativeof the abundance of target binding partners in proximity to each otherwithin the sample. 13-26. (canceled)
 27. A method for treating amalignancy in a subject, comprising administering a protein-proteininteraction (PPI) modulator to the subject, wherein the subject ispredetermined to be sensitive to the PPI modulator based on a PPIprofile obtained from a sample of the malignancy.
 28. A method fortreating a malignancy in a subject, comprising: (a) assessing thesensitivity of a malignancy in the subject, comprising comparing aprotein-protein interaction (PPI) profile obtained from a sample of themalignancy to a reference PPI profile; and (b) administering a PPImodulator to the subject if the malignancy is assessed to be sensitiveto the PPI modulator; and withholding the PPI modulator from the subjectif the malignancy is assessed to be resistant to the PPI modulator. 29.A kit for quantitatively detecting the proximity of target bindingpartners within a cancer protein-protein interaction network, comprisinga primary antibody to at least one of the target binding partners; and aproximity probe comprising a secondary antibody (that binds to theprimary antibody) with an oligonucleotide conjugated thereto.
 30. Thekit of claim 29, wherein the kit comprises: a first primary antibody toa first target binding partner; a second primary antibody to a secondtarget binding partner; a first proximity probe comprising a firstsecondary antibody (that binds to the first primary antibody) with anoligonucleotide conjugated thereto; and a second proximity probecomprising a second secondary antibody (that binds to the second primaryantibody) with an oligonucleotide conjugated thereto, wherein when theoligonucleotides of the first and second proximity probes are insufficient proximity to each other, the oligonucleotides of theproximity probes interact in the presence circle-formingoligonucleotides by enzymatic ligation and form a circular product thatis amplified by rolling circle replication, producing an amplificationproduct. 31-37. (canceled)
 38. A kit for quantitatively detecting theproximity of target binding partners within a tertiary interaction (aninteraction of three or more target binding partners), comprising: afirst primary antibody to a first target binding partner of the tertiaryinteraction; a second primary antibody to a second target bindingpartner of the tertiary interaction; a third primary antibody to a thirdtarget binding partner of the tertiary interaction; a first proximityprobe comprising a first secondary antibody (that binds to the firstprimary antibody) with an oligonucleotide conjugated thereto; and asecond proximity probe comprising a second secondary antibody (thatbinds to the second primary antibody) with an oligonucleotide conjugatedthereto; a third proximity probe comprising a third secondary antibody(that binds to the third primary antibody) with an oligonucleotideconjugated thereto; wherein when the oligonucleotides of the first andsecond proximity probes are in sufficient proximity to each other, theoligonucleotides of the first and second proximity probes interact inthe presence circle-forming oligonucleotides by enzymatic ligation andform a circular product that is amplified by rolling circle replication,producing a first amplification product; and wherein when theoligonucleotides of the second and third proximity probes are insufficient proximity to each other, the oligonucleotides of the secondand third proximity probes interact in the presence of circle-formingoligonucleotides by enzymatic ligation and form a circular product thatis amplified by rolling circle replication, producing a secondamplification product.
 39. The kit of claim 38, wherein the kit furthercomprises: a first labeled oligonucleotide probe that hybridizes withthe first amplification product, allowing detection and quantificationof the first amplification product (representing the association (closeproximity) of the first and second target binding partners); and asecond labeled oligonucleotide probe that hybridizes with the secondamplification product, allowing detection and quantification of thesecond amplification product (representing the association of the secondand third target binding partners), wherein the label of the firstlabeled oligonucleotide yields a signal that is distinguishable from thesignal of the second labeled oligonucleotide. 40-46. (canceled)
 47. Amethod for measuring protein-protein interactions (PPIs) having three ormore binding partners (a tertiary interaction) in a sample, comprising:contacting the sample with three or more primary antibodies to three ormore corresponding target binding partners within a target tertiaryinteraction; contacting the sample with three or more proximity probes(first, second, and third proximity probes) comprising a secondaryantibody that binds to the corresponding first antibody, wherein eachproximity probe has an oligonucleotide conjugated thereto; wherein whenthe oligonucleotides of the first and second proximity probes are insufficient proximity to each other, the oligonucleotides of the firstand second proximity probes interact in the presence circle-formingoligonucleotides by enzymatic ligation and form a circular product thatis amplified by rolling circle replication, producing a firstamplification product; and wherein when the oligonucleotides of thesecond and third proximity probes are in sufficient proximity to eachother, the oligonucleotides of the second and third proximity probesinteract in the presence of circle-forming oligonucleotides by enzymaticligation and form a circular product that is amplified by rolling circlereplication, producing a second amplification product; and measuring thefirst and second amplification products.
 48. The method of claim 47,wherein said measuring comprises: contacting the sample with two or morelabeled oligonucleotides, comprising a first labeled oligonucleotidethat hybridizes to a sequence of the first amplification product, and asecond labeled oligonucleotide that hybridizes to a sequence of thesecond amplification product, to produce labeled amplification products,wherein the labels are distinguishable from one another; and measuringthe PPIs by visualizing the labeled amplification products. 49-55.(canceled)
 56. A method for the identification of a biomarker,comprising selecting two or more target binding partners within a cancersample; generating a PPI profile for the two or more target bindingpartners; comparing the PPI profile to the responsiveness of the cancerto a treatment in vitro and/or in vivo (for example, in xenograft modelsor human subjects).
 57. The method of claim 56, wherein the treatment isa kinase inhibitor. 58-63. (canceled)
 64. A method for identifying anagent as a protein-protein interaction (PPI) modulator, comprising:contacting cancer cells with a candidate agent in vitro or in vivo; anddetermining whether the candidate agent modulates a selected PPI in asample of the cancer cells.
 65. The method of claim 64, wherein saiddetermining comprises comparing a first PPI profile of the cancer cellsobtained prior to said contacting to a second PPI profile of the cancercells obtained after said contacting, wherein a change in the PPI isindicative of a PPI modulator. 66-72. (canceled)